Compositions and methods for the depletion of cells

ABSTRACT

The invention provides compositions and methods useful for the depletion of cells, such as CD45+, CD135+, CD34+, CD90+, and/or CD110+ cells, and for the treatment of various hematopoietic diseases, metabolic disorders, cancers, and autoimmune diseases, among others. Described herein are antibodies, antigen-binding fragments, ligands, and conjugates thereof that can be applied to effect the treatment of these conditions, for instance, by depleting a population of CD45+, CD135+, CD34+, CD90+, or CD110+ cells in a patient, such as a human. The compositions and methods described herein can be used to treat a disorder directly, for instance, by depleting a population of CD45+, CD135+, CD34+, CD90+, or CD110+ cancer cells or autoimmune cells. The compositions and methods described herein can also be used to prepare a patient for hematopoietic stem cell transplant therapy and to improve the engraftment of hematopoietic stem cell transplants by selectively depleting endogenous hematopoietic stem cells prior to the transplant procedure.

FIELD OF THE INVENTION

The invention relates to the treatment of patients suffering fromvarious pathologies, such as blood diseases, metabolic disorders,cancers, and autoimmune diseases, among others, by administration of anantibody, antigen-binding fragment thereof, or ligand capable of bindingan antigen expressed by a hematopoietic cell, such as a hematopoieticstem cell.

BACKGROUND OF THE INVENTION

Despite advances in the medicinal arts, there remains a demand fortreating pathologies of the hematopoietic system, such as diseases of aparticular blood cell, metabolic disorders, cancers, and autoimmuneconditions, among others. While hematopoietic stem cells havesignificant therapeutic potential, a limitation that has hindered theiruse in the clinic has been the difficulty associated with ensuringengraftment of hematopoietic stem cell transplants in a host. There iscurrently a need for compositions and methods for promoting theengraftment of exogenous hematopoietic stem cell grafts such that themulti-potency and hematopoietic functionality of these cells ispreserved following transplantation.

SUMMARY OF THE INVENTION

The present invention provides compositions and methods for the directtreatment of various disorders of the hematopoietic system, metabolicdisorders, cancers, and autoimmune diseases, among others. The inventionadditionally features methods for conditioning a patient, such as ahuman patient, prior to receiving hematopoietic stem cell transplanttherapy so as to promote the engraftment of hematopoietic stem cellgrafts. The patient may be one that is suffering from one or more blooddisorders, such as a hemoglobinopathy or other hematopoietic pathology,and is thus in need of hematopoietic stem cell transplantation. Asdescribed herein, hematopoietic stem cells are capable ofdifferentiating into a multitude of cell types in the hematopoieticlineage, and can be administered to a patient in order to populate orre-populate a cell type that is deficient in the patient. The inventionfeatures methods of treating a patient with antibodies, antibodyfragments, ligands, drug-antibody conjugates, and drug-ligand conjugatescapable of binding proteins expressed by hematopoietic cells, such ascell-surface hematopoietic cell antigens, so as to (i) directly treat adisease such as a blood disorder, metabolic disease, cancer, orautoimmune disease, among others described herein, by selectivelydepleting a population of cells that express CD45, CD135, CD34, CD90,and/or CD110, such as an aberrant blood cell, cancer cell, or autoimmunecell, and/or (ii) deplete a population of endogenous hematopoietic stemcells within the patient. The former activity enables the directtreatment of a wide range of disorders associated with a cell of thehematopoietic lineage, as CD45, CD135, CD34, CD90, and/or CD110 may beexpressed by a cancerous cell, such as a leukemic cell, an autoimmunelymphocyte, such as a T-cell that expresses a T-cell receptor thatcross-reacts with a self antigen, among other cell types. The latteractivity, the selective depletion of hematopoietic stem cells, in turncreates a vacancy that can subsequently be filled by transplantation ofan exogenous (for instance, an autologous, allogeneic, or syngeneic)hematopoietic stem cell graft. The invention thus provides methods oftreating a variety of hematopoietic conditions, such as sickle cellanemia, thalassemia, Fanconi anemia, Wiskott-Aldrich syndrome, adenosinedeaminase deficiency-severe combined immunodeficiency, metachromaticleukodystrophy, Diamond-Blackfan anemia and Schwachman-Diamond syndrome,human immunodeficiency virus infection, and acquired immune deficiencysyndrome, as well as cancers and autoimmune diseases, among others.

In a first aspect, the invention provides a method of depleting apopulation of CD135+ cells in a human patient by administering aneffective amount of an antibody or antigen-binding fragment thereofcapable of binding CD135 conjugated to a cytotoxin.

In another aspect, the invention provides a method of depleting apopulation of CD135+ cells in a human patient in need of a hematopoieticstem cell transplant by administering, prior to the patient receiving atransplant containing hematopoietic stem cells, an effective amount ofan antibody or antigen-binding fragment thereof capable of binding CD135conjugated to a cytotoxin.

In an additional aspect, the invention features a method, for example,of treating a human patient in need of a hematopoietic stem celltransplant, including administering to a human patient a transplantcontaining hematopoietic stem cells, wherein the patient has beenpreviously administered an antibody or antigen-binding fragment thereofcapable of binding CD135 conjugated to a cytotoxin in an amountsufficient to deplete a population of CD135+ cells in the patient.

In another aspect, the invention features a method, for example, oftreating a human patient in need of a hematopoietic stem celltransplant, including: administering to a human patient an antibody orantigen-binding fragment thereof capable of binding CD135 conjugated toa cytotoxin in an amount sufficient to deplete a population of CD135+cells in the patient, and subsequently administering to the patient atransplant containing hematopoietic stem cells.

In some embodiments of the four preceding aspects, the antibody orantigen-binding fragment thereof contains the following complementaritydetermining regions (CDRs):

(SEQ ID NO: 1) a CDR-H1 having the amino acid sequence SYYMH;(SEQ ID NO: 2) a CDR-H2 having the amino acid sequenceIINPSGGSTSYAQKFQG; (SEQ ID NO: 3)a CDR-H3 having the amino acid sequence GVGAHDAFDI or (SEQ ID NO: 4)VVAAAVADY; (SEQ ID NO: 5) a CDR-L1 having the amino acid sequenceRSSQSLLHSNGNNYLD or (SEQ ID NO: 6) RSSQSLLHSNGYNYLD; (SEQ ID NO: 7)a CDR-L2 having the amino acid sequence LGSNRAS; and (SEQ ID NO: 8)a CDR-L3 having the amino acid sequence MQGTHPAIS or (SEQ ID NO: 9)MQSLQTPFT.

In some embodiments of the four preceding aspects, the antibody orantigen-binding fragment thereof contains the following CDRs:

(SEQ ID NO: 10) a CDR-H1 having the amino acid sequence SYAIS;(SEQ ID NO: 11) a CDR-H2 having the amino acid sequenceGIIPIFGTANYAQKFQG; (SEQ ID NO: 12)a CDR-H3 having the amino acid sequence FALFGFREQAFDI; (SEQ ID NO: 13)a CDR-L1 having the amino acid sequence RASQSISSYLN; (SEQ ID NO: 14)a CDR-L2 having the amino acid sequence AASSLQS; and (SEQ ID NO: 15)a CDR-L3 having the amino acid sequence QQSYSTPFT.

In another aspect, the invention features a method of depleting apopulation of CD135+ cells in a human patient by administering to thepatient an effective amount of human Flt3 ligand, or a fragment thereofcapable of binding CD135.

In another aspect, the invention features a method of depleting apopulation of CD135+ cells in a human patient in need of a hematopoieticstem cell transplant by administering to the patient an effective amountof human Flt3 ligand, or a fragment thereof capable of binding CD135,prior to the patient receiving a transplant containing hematopoieticstem cells.

In another aspect, the invention features a method, for example, oftreating a human patient in need of a hematopoietic stem celltransplant, including administering to a human patient a transplantcontaining hematopoietic stem cells, wherein the patient has beenpreviously administered human Flt3 ligand, or a fragment thereof capableof binding CD135, in an amount sufficient to deplete a population ofCD135+ cells in the patient.

In an additional aspect, the invention features a method, for example,of treating a human patient in need of a hematopoietic stem celltransplant, including administering to the patient human Flt3 ligand, ora fragment thereof capable of binding CD135, in an amount sufficient todeplete a population of CD135+ cells in the patient; and subsequentlyadministering to the patient a transplant containing hematopoietic stemcells.

In some embodiments of any of the preceding four aspects, the human Flt3ligand or fragment thereof is covalently bound to an Fc domain, such asa dimeric Fc domain isolated from a human antibody (for example,isolated from an IgG1, IgG2, IgG3, or IgG4 isotype human antibody). Insome embodiments, the Fc domain is a monomeric Fc domain containing asingle polypeptide strand. In some embodiments, the N-terminus of thehuman Flt3 ligand or fragment thereof is bound to the Fc domain. In someembodiments, the C-terminus of the human Flt3 ligand or fragment thereofis bound to the Fc domain. The Fc domain may be conjugated to one ormore copies of the Flt3 ligand or fragment thereof. For instance,conjugates that may be used with the methods described herein includedimeric Fc domains in which each polypeptide strand of the Fc domain isconjugated to a human Flt3 ligand or fragment thereof. The Fc domain mayin turn be conjugated to a cytotoxin, such as a cytotoxin describedherein (for example, pseudomonas exotoxin A, deBouganin, diphtheriatoxin, an amatoxin, such as α-amanitin, saporin, maytansine, amaytansinoid, an auristatin, an anthracycline, a calicheamicin,irinotecan, SN-38, a duocarmycin, a pyrrolobenzodiazepine, apyrrolobenzodiazepine dimer, an indolinobenzodiazepine, and anindolinobenzodiazepine dimer, or a variant thereof).

In some embodiments of any of the preceding four aspects, the human Flt3ligand or fragment thereof is covalently bound to a cytotoxin, such as acytotoxin described herein (for example, pseudomonas exotoxin A,deBouganin, diphtheria toxin, an amatoxin, such as α-amanitin, saporin,maytansine, a maytansinoid, an auristatin, an anthracycline, acalicheamicin, irinotecan, SN-38, a duocarmycin, apyrrolobenzodiazepine, a pyrrolobenzodiazepine dimer, anindolinobenzodiazepine, and an indolinobenzodiazepine dimer, or avariant thereof). In some embodiments, the N-terminus of the human Flt3ligand or fragment thereof is bound to the cytotoxin. In someembodiments, the C-terminus of the human Flt3 ligand or fragment thereofis bound to the cytotoxin. The cytotoxin may in turn be conjugated to anFc domain.

In some embodiments of any of the preceding four aspects, the human Flt3ligand or fragment thereof is covalently bound to the cytotoxin at onesite on the human Flt3 ligand or fragment thereof (for example, the N-or C-terminus of the human Flt3 ligand or fragment thereof) and iscovalently bound to an Fc domain at another site on the human Flt3ligand or fragment thereof (for example, the opposite terminus of thehuman Flt3 ligand or fragment thereof).

In some embodiments of any of the preceding four aspects, the Fc domainis a human IgG1 isotype Fc domain. In some embodiments, the Fc domain isa human IgG2 isotype Fc domain. In some embodiments, the Fc domain is ahuman IgG3 isotype Fc domain. In some embodiments, the Fc domain is ahuman IgG4 isotype Fc domain.

In another aspect, the invention features a method of depleting apopulation of CD34+ cells in a human patient by administering aneffective amount of an antibody or antigen-binding fragment thereofcapable of binding CD34 conjugated to a cytotoxin.

In another aspect, the invention features a method of depleting apopulation of CD34+ cells in a human patient in need of a hematopoieticstem cell transplant by administering, prior to the patient receiving atransplant including hematopoietic stem cells, an effective amount of anantibody or antigen-binding fragment thereof capable of binding CD34conjugated to a cytotoxin.

In another aspect, the invention provides a method, for example, oftreating a human patient in need of a hematopoietic stem celltransplant, including administering to a human patient a transplantincluding hematopoietic stem cells, wherein the patient has beenpreviously administered an antibody or antigen-binding fragment thereofcapable of binding CD34 conjugated to a cytotoxin in an amountsufficient to deplete a population of CD34+ cells in the patient.

In an additional aspect, the invention features a method, for example,of treating a human patient in need of a hematopoietic stem celltransplant, including: administering to the patient an antibody orantigen-binding fragment thereof capable of binding CD34 conjugated to acytotoxin in an amount sufficient to deplete a population of CD34+ cellsin the patient, and subsequently administering to the patient atransplant including hematopoietic stem cells.

In another aspect, the invention features a method of depleting apopulation of CD90+ cells in a human patient by administering aneffective amount of an antibody or antigen-binding fragment thereofcapable of binding CD90 conjugated to a cytotoxin.

In another aspect, the invention features a method of depleting apopulation of CD90+ cells in a human patient in need of a hematopoieticstem cell transplant by administering, prior to the patient receiving atransplant including hematopoietic stem cells, an effective amount of anantibody or antigen-binding fragment thereof capable of binding CD90conjugated to a cytotoxin.

In another aspect, the invention provides a method, for example, oftreating a human patient in need of a hematopoietic stem celltransplant, including administering to a human patient a transplantincluding hematopoietic stem cells, wherein the patient has beenpreviously administered an antibody or antigen-binding fragment thereofcapable of binding CD90 conjugated to a cytotoxin in an amountsufficient to deplete a population of CD90+ cells in the patient.

In an additional aspect, the invention features a method, for example,of treating a human patient in need of a hematopoietic stem celltransplant, including: administering to the patient an antibody orantigen-binding fragment thereof capable of binding CD90 conjugated to acytotoxin in an amount sufficient to deplete a population of CD90+ cellsin the patient, and subsequently administering to the patient atransplant including hematopoietic stem cells.

In another aspect, the invention features a method of depleting apopulation of CD110+ cells in a human patient by administering aneffective amount of an antibody or antigen-binding fragment thereofcapable of binding CD110 conjugated to a cytotoxin.

In another aspect, the invention features a method of depleting apopulation of CD110+ cells in a human patient in need of a hematopoieticstem cell transplant by administering, prior to the patient receiving atransplant including hematopoietic stem cells, an effective amount of anantibody or antigen-binding fragment thereof capable of binding CD110conjugated to a cytotoxin.

In another aspect, the invention provides a method, for example, oftreating a human patient in need of a hematopoietic stem celltransplant, including administering to the patient a transplantincluding hematopoietic stem cells, wherein the patient has beenpreviously administered an antibody or antigen-binding fragment thereofcapable of binding CD110 conjugated to a cytotoxin in an amountsufficient to deplete a population of CD110+ cells in the patient.

In an additional aspect, the invention features a method, for example,of treating a human patient in need of a hematopoietic stem celltransplant, including: administering to the patient an antibody orantigen-binding fragment thereof capable of binding CD110 conjugated toa cytotoxin in an amount sufficient to deplete a population of CD110+cells in the patient, and subsequently administering to the patient atransplant including hematopoietic stem cells.

In some embodiments of any of the foregoing aspects, the cytotoxinconjugated to the antibody, antigen-binding fragment thereof, or ligandis pseudomonas exotoxin A, deBouganin, diphtheria toxin, an amatoxin,such as α-amanitin, saporin, maytansine, a maytansinoid, an auristatin,an anthracycline, a calicheamicin, irinotecan, SN-38, a duocarmycin, apyrrolobenzodiazepine, a pyrrolobenzodiazepine dimer, anindolinobenzodiazepine, or an indolinobenzodiazepine dimer, or a variantthereof.

In another aspect, the invention provides a method of depleting apopulation of CD45+ cells in a human patient by administering aneffective amount of an antibody or antigen-binding fragment thereofcapable of binding CD45 conjugated to a cytotoxin. The cytotoxin may be,for example, pseudomonas exotoxin A, deBouganin, diphtheria toxin, anamatoxin, such as α-amanitin, saporin, maytansine, a maytansinoid, anauristatin, an anthracycline, a calicheamicin, irinotecan, SN-38, aduocarmycin, a pyrrolobenzodiazepine, a pyrrolobenzodiazepine dimer, anindolinobenzodiazepine, or an indolinobenzodiazepine dimer, or a variantthereof.

In another aspect, the invention provides a method of depleting apopulation of CD45+ cells in a human patient in need of a hematopoieticstem cell transplant by administering, prior to the patient receiving atransplant including hematopoietic stem cells, an effective amount of anantibody or antigen-binding fragment thereof capable of binding CD45conjugated to a cytotoxin. The cytotoxin may be, for example,pseudomonas exotoxin A, deBouganin, diphtheria toxin, an amatoxin, suchas α-amanitin, saporin, maytansine, a maytansinoid, an auristatin, ananthracycline, a calicheamicin, irinotecan, SN-38, a duocarmycin, apyrrolobenzodiazepine, a pyrrolobenzodiazepine dimer, anindolinobenzodiazepine, or an indolinobenzodiazepine dimer, or a variantthereof.

In another aspect, the invention features a method, for example, oftreating a human patient in need of a hematopoietic stem celltransplant, including administering to a human patient a transplantincluding hematopoietic stem cells, wherein the patient has beenpreviously administered an antibody or antigen-binding fragment thereofcapable of binding CD45 conjugated to a cytotoxin in an amountsufficient to deplete a population of CD45+ cells in the patient. Thecytotoxin may be, for example, pseudomonas exotoxin A, deBouganin,diphtheria toxin, an amatoxin, such as α-amanitin, saporin, maytansine,a maytansinoid, an auristatin, an anthracycline, a calicheamicin,irinotecan, SN-38, a duocarmycin, a pyrrolobenzodiazepine, apyrrolobenzodiazepine dimer, an indolinobenzodiazepine, or anindolinobenzodiazepine dimer, or a variant thereof

In an additional aspect, the invention features a method, for example,of treating a human patient in need of a hematopoietic stem celltransplant, including: administering to a human patient an antibody orantigen-binding fragment thereof capable of binding CD45 conjugated to acytotoxin, such as pseudomonas exotoxin A, deBouganin, diphtheria toxin,an amatoxin, such as α-amanitin, saporin, maytansine, a maytansinoid, anauristatin, an anthracycline, a calicheamicin, irinotecan, SN-38, aduocarmycin, a pyrrolobenzodiazepine, a pyrrolobenzodiazepine dimer, anindolinobenzodiazepine, or an indolinobenzodiazepine dimer, or a variantthereof, in an amount sufficient to deplete a population of CD45+ cellsin the patient, and subsequently administering to the patient atransplant including hematopoietic stem cells.

In some embodiments of the preceding four aspects, the CD45 is CD45RO.

In another aspect, the invention provides a method of depleting apopulation of CD45RO+ cells in a human patient by administering aneffective amount of an antibody or antigen-binding fragment thereofcapable of binding CD45RO.

In another aspect, the invention provides a method of depleting apopulation of CD45RO+ cells in a human patient in need of ahematopoietic stem cell transplant by administering, prior to thepatient receiving a transplant containing hematopoietic stem cells, aneffective amount of an antibody or antigen-binding fragment thereofcapable of binding CD45RO.

In another aspect, the invention features a method, for example, oftreating a human patient in need of a hematopoietic stem celltransplant, including administering to a human patient a transplantincluding hematopoietic stem cells, wherein the patient has beenpreviously administered an antibody or antigen-binding fragment thereofcapable of binding CD45RO in an amount sufficient to deplete apopulation of CD45RO+ cells in the patient.

In an additional aspect, the invention features a method, for example,of treating a human patient in need of a hematopoietic stem celltransplant, including: administering to the patient an antibody orantigen-binding fragment thereof capable of binding CD45RO in an amountsufficient to deplete a population of CD45RO+ cells in the patient, andsubsequently administering to the patient a transplant includinghematopoietic stem cells.

In some embodiments of any of the preceding four aspects of theinvention, the antibody or antigen-binding fragment thereof isconjugated to a cytotoxin.

In another aspect, the invention provides a method of depleting apopulation of CD45+ cells in a human patient by administering aneffective amount of a ligand or fragment thereof capable of bindingCD45.

In another aspect, the invention provides a method of depleting apopulation of CD45+ cells in a human patient in need of a hematopoieticstem cell transplant by administering, prior to the patient receiving atransplant including hematopoietic stem cells, an effective amount of aligand or fragment thereof capable of binding CD45.

In another aspect, the invention features a method, for example, oftreating a human patient in need of a hematopoietic stem celltransplant, including administering to a human patient a transplantincluding hematopoietic stem cells, wherein the patient has beenpreviously administered a ligand or fragment thereof capable of bindingCD45 in an amount sufficient to deplete a population of CD45+ cells inthe patient.

In an additional aspect, the invention features a method, for example,of treating a human patient in need of a hematopoietic stem celltransplant, including: administering to a human patient a ligand orfragment thereof capable of binding CD45 in an amount sufficient todeplete a population of CD45+ cells in the patient, and subsequentlyadministering to the patient a transplant including hematopoietic stemcells.

In another aspect, the invention provides a method of depleting apopulation of CD34+ cells in a human patient by administering aneffective amount of a ligand or fragment thereof capable of bindingCD34.

In another aspect, the invention provides a method of depleting apopulation of CD34+ cells in a human patient in need of a hematopoieticstem cell transplant by administering, prior to the patient receiving atransplant including hematopoietic stem cells, an effective amount of aligand or fragment thereof capable of binding CD34.

In another aspect, the invention features a method, for example, oftreating a human patient in need of a hematopoietic stem celltransplant, including administering to a human patient a transplantincluding hematopoietic stem cells, wherein the patient has beenpreviously administered a ligand or fragment thereof capable of bindingCD34 in an amount sufficient to deplete a population of CD34+ cells inthe patient.

In an additional aspect, the invention features a method, for example,of treating a human patient in need of a hematopoietic stem celltransplant, including: administering to a human patient a ligand orfragment thereof capable of binding CD34 in an amount sufficient todeplete a population of CD34+ cells in the patient, and subsequentlyadministering to the patient a transplant including hematopoietic stemcells.

In another aspect, the invention provides a method of depleting apopulation of CD90+ cells in a human patient by administering aneffective amount of a ligand or fragment thereof capable of bindingCD90.

In another aspect, the invention provides a method of depleting apopulation of CD90+ cells in a human patient in need of a hematopoieticstem cell transplant by administering, prior to the patient receiving atransplant including hematopoietic stem cells, an effective amount of aligand or fragment thereof capable of binding CD90.

In another aspect, the invention features a method, for example, oftreating a human patient in need of a hematopoietic stem celltransplant, including administering to a human patient a transplantincluding hematopoietic stem cells, wherein the patient has beenpreviously administered a ligand or fragment thereof capable of bindingCD90 in an amount sufficient to deplete a population of CD90+ cells inthe patient.

In an additional aspect, the invention features a method, for example,of treating a human patient in need of a hematopoietic stem celltransplant, including: administering to a human patient a ligand orfragment thereof capable of binding CD90 in an amount sufficient todeplete a population of CD90+ cells in the patient, and subsequentlyadministering to the patient a transplant including hematopoietic stemcells.

In another aspect, the invention provides a method of depleting apopulation of CD110+ cells in a human patient by administering aneffective amount of a ligand or fragment thereof capable of bindingCD110.

In another aspect, the invention provides a method of depleting apopulation of CD110+ cells in a human patient in need of a hematopoieticstem cell transplant by administering, prior to the patient receiving atransplant including hematopoietic stem cells, an effective amount of aligand or fragment thereof capable of binding CD110.

In another aspect, the invention features a method, for example, oftreating a human patient in need of a hematopoietic stem celltransplant, including administering to a human patient a transplantincluding hematopoietic stem cells, wherein the patient has beenpreviously administered a ligand or fragment thereof capable of bindingCD110 in an amount sufficient to deplete a population of CD110+ cells inthe patient.

In an additional aspect, the invention features a method, for example,of treating a human patient in need of a hematopoietic stem celltransplant, including: administering to a human patient a ligand orfragment thereof capable of binding CD110 in an amount sufficient todeplete a population of CD110+ cells in the patient, and subsequentlyadministering to the patient a transplant including hematopoietic stemcells.

In some embodiments of the foregoing aspects, the ligand or fragmentthereof that binds one or more of CD45 (e.g., CD45RO), CD34, CD90, andCD110 is covalently bound to an Fc domain, such as a dimeric Fc domainisolated from a human antibody (for example, isolated from an IgG1,IgG2, IgG3, or

IgG4 isotype human antibody). In some embodiments, the Fc domain is amonomeric Fc domain containing a single polypeptide strand. In someembodiments, the N-terminus of the ligand or fragment thereof is boundto the Fc domain. In some embodiments, the C-terminus of the ligand orfragment thereof is bound to the Fc domain. The Fc domain may beconjugated to one or more copies of the ligand or fragment thereof. Forinstance, conjugates that may be used with the methods described hereininclude dimeric Fc domains in which each polypeptide strand of the Fcdomain is conjugated to the ligand or fragment thereof. The Fc domainmay in turn be conjugated to a cytotoxin, such as a cytotoxin describedherein (for example, pseudomonas exotoxin A, deBouganin, diphtheriatoxin, an amatoxin, such as α-amanitin, saporin, maytansine, amaytansinoid, an auristatin, an anthracycline, a calicheamicin,irinotecan, SN-38, a duocarmycin, a pyrrolobenzodiazepine, apyrrolobenzodiazepine dimer, an indolinobenzodiazepine, and anindolinobenzodiazepine dimer, or a variant thereof).

In some embodiments of any of the foregoing aspects, the ligand orfragment thereof is covalently bound to a cytotoxin, such as a cytotoxindescribed herein (for example, pseudomonas exotoxin A, deBouganin,diphtheria toxin, an amatoxin, such as α-amanitin, saporin, maytansine,a maytansinoid, an auristatin, an anthracycline, a calicheamicin,irinotecan, SN-38, a duocarmycin, a pyrrolobenzodiazepine, apyrrolobenzodiazepine dimer, an indolinobenzodiazepine, and anindolinobenzodiazepine dimer, or a variant thereof). In someembodiments, the N-terminus of the ligand or fragment thereof is boundto the cytotoxin. In some embodiments, the C-terminus of the ligand orfragment thereof is bound to the cytotoxin. The cytotoxin may in turn beconjugated to an Fc domain.

In some embodiments of any of the foregoing aspects, the ligand orfragment thereof is covalently bound to the cytotoxin at one site on theligand or fragment thereof (for example, the N- or C-terminus of theligand or fragment thereof) and is covalently bound to an Fc domain atanother site on the ligand or fragment thereof (for example, theopposite terminus of the ligand or fragment thereof).

In some embodiments of any of the foregoing aspects, the Fc domain is ahuman IgG1 isotype Fc domain. In some embodiments, the Fc domain is ahuman IgG2 isotype Fc domain. In some embodiments, the Fc domain is ahuman IgG3 isotype Fc domain. In some embodiments, the Fc domain is ahuman IgG4 isotype Fc domain.

In some embodiments of any of the above aspects, the cytotoxin is anamatoxin or derivative thereof, such as α-amanitin, β-amanitin,γ-amanitin, ε-amanitin, amanin, amaninamide, amanullin, amanullinicacid, and proamanullin. In some embodiments of any of the above aspects,the cytotoxin is an amatoxin, and the antibody, antigen-binding fragmentthereof, or ligand conjugated to the cytotoxin is represented by theformula Ab-Am, wherein Ab is the antibody, antigen-binding fragmentthereof, or ligand, and Am is an amatoxin. In some embodiments, Am isrepresented by formula (I)

wherein R₁ is H, OH, OR_(A), or OR_(C);

R₂ is H, OH, OR_(B), or OR_(C);

R_(A) and R_(B), together with the oxygen atoms to which they are bound,combine to form an optionally substituted 5-membered heterocyclolalkylgroup;

R₃ is H, R_(C), or R_(D);

R₄ is H, OH, OR_(C), OR_(D), R_(C), or R_(D);

R₅ is H, OH, OR_(C), OR_(D), R_(C), or R_(D);

R₆ is H, OH, OR_(C), OR_(D), R_(C), or R_(D);

R₇ is H, OH, OR_(C), OR_(D), R_(C), or R_(D);

R₈ is OH, NH₂, OR_(C), OR_(D), NHR_(C), or NR_(C)R_(D);

R₉ is H, OH, OR_(C), or OR_(D);

X is —S—, —S(O)—, or —SO₂—;

R_(C) is -L-Z;

R_(D) is optionally substituted alkyl (e.g., C₁-C₆ alkyl), optionallysubstituted heteroalkyl (e.g., C₁-C₆ heteroalkyl), optionallysubstituted alkenyl (e.g., C₂-C₆ alkenyl), optionally substitutedheteroalkenyl (e.g., C₂-C₆ heteroalkenyl), optionally substitutedalkynyl (e.g., C₂-C₆ alkynyl), optionally substituted heteroalkynyl(e.g., C₂-C₆ heteroalkynyl), optionally substituted cycloalkyl,optionally substituted heterocycloalkyl, optionally substituted aryl, oroptionally substituted heteroaryl;

L is a linker, such as optionally substituted alkylene (e.g., C₁-C₆alkylene), optionally substituted heteroalkylene (C₁-C₆ heteroalkylene),optionally substituted alkenylene (e.g., C₂-C₆ alkenylene), optionallysubstituted heteroalkenylene (e.g., C₂-C₆ heteroalkenylene), optionallysubstituted alkynylene (e.g., C₂-C₆ alkynylene), optionally substitutedheteroalkynylene (e.g., C₂-C₆ heteroalkynylene), optionally substitutedcycloalkylene, optionally substituted heterocycloalkylene, optionallysubstituted arylene, or optionally substituted heteroarylene; and

Z is a chemical moiety formed from a coupling reaction between areactive substituent present on L and a reactive substituent presentwithin an antibody, antigen-binding fragment thereof, or ligand thatbinds CD45 (such as CD45RO), CD135, CD34, CD90, and/or CD110.

In some embodiments, Am contains exactly one R_(C) substituent.

In some embodiments, Am is represented by formula (IA)

wherein R₁ is H, OH, OR_(A), or OR_(C);

R₂ is H, OH, OR_(B), or OR_(C);

R_(A) and R_(B), together with the oxygen atoms to which they are bound,combine to form an optionally substituted 5-membered heterocyclolalkylgroup;

R₃ is H, R_(C), or R_(D);

R₄ is H, OH, OR_(C), OR_(D), R_(C), or R_(D);

R₅ is H, OH, OR_(C), OR_(D), R_(C), or R_(D);

R₆ is H, OH, OR_(C), OR_(D), R_(C), or R_(D);

R₇ is H, OH, OR_(C), OR_(D), R_(C), or R_(D);

R₈ is OH, NH₂, OR_(C), OR_(D), NHR_(C), or NR_(C)R_(D);

R₉ is H, OH, OR_(C), or OR_(D);

X is —S—, —S(O)—, or —SO₂—;

R_(C) is -L-Z;

R_(D) is optionally substituted alkyl (e.g., C₁-C₆ alkyl), optionallysubstituted heteroalkyl (e.g., C₁-C₆ heteroalkyl), optionallysubstituted alkenyl (e.g., C₂-C₆ alkenyl), optionally substitutedheteroalkenyl (e.g., C₂-C₆ heteroalkenyl), optionally substitutedalkynyl (e.g., C₂-C₆ alkynyl), optionally substituted heteroalkynyl(e.g., C₂-C₆ heteroalkynyl), optionally substituted cycloalkyl,optionally substituted heterocycloalkyl, optionally substituted aryl, oroptionally substituted heteroaryl; L is a linker, such as optionallysubstituted alkylene (e.g., C₁-C₆ alkylene), optionally substitutedheteroalkylene (C₁-C₆ heteroalkylene), optionally substituted alkenylene(e.g., C₂-C₆ alkenylene), optionally substituted heteroalkenylene (e.g.,C₂-C₆ heteroalkenylene), optionally substituted alkynylene (e.g., C₂-C₆alkynylene), optionally substituted heteroalkynylene (e.g., C₂-C₆heteroalkynylene), optionally substituted cycloalkylene, optionallysubstituted heterocycloalkylene, optionally substituted arylene, oroptionally substituted heteroarylene;

Z is a chemical moiety formed from a coupling reaction between areactive substituent present on L and a reactive substituent presentwithin an antibody, antigen-binding fragment thereof, or ligand thatbinds CD45 (such as CD45RO), CD135, CD34, CD90, and/or CD110; and

wherein Am contains exactly one R_(C) substituent.

In some embodiments, Am is represented by formula (IB)

wherein R₁ is H, OH, OR_(A), or OR_(C);

R₂ is H, OH, OR_(B), or OR_(C);

R_(A) and R_(B), together with the oxygen atoms to which they are bound,combine to form an optionally substituted 5-membered heterocyclolalkylgroup;

R₃ is H, R_(C), or R_(D);

R₄ is H, OH, OR_(C), OR_(D), R_(C), or R_(D);

R₅ is H, OH, OR_(C), OR_(D), R_(C), or R_(D);

R₆ is H, OH, OR_(C), OR_(D), R_(C), or R_(D);

R₇ is H, OH, OR_(C), OR_(D), R_(C), or R_(D);

R₈ is OH, NH₂, OR_(C), OR_(D), NHR_(C), or NR_(C)R_(D);

R₉ is H, OH, OR_(C), or OR_(D);

X is —S—, —S(O)—, or —SO₂—;

R_(C) is -L-Z;

R_(D) is optionally substituted alkyl (e.g., C₁-C₆ alkyl), optionallysubstituted heteroalkyl (e.g., C₁-C₆ heteroalkyl), optionallysubstituted alkenyl (e.g., C₂-C₆ alkenyl), optionally substitutedheteroalkenyl (e.g., C₂-C₆ heteroalkenyl), optionally substitutedalkynyl (e.g., C₂-C₆ alkynyl), optionally substituted heteroalkynyl(e.g., C₂-C₆ heteroalkynyl), optionally substituted cycloalkyl,optionally substituted heterocycloalkyl, optionally substituted aryl, oroptionally substituted heteroaryl;

L is a linker, such as optionally substituted alkylene (e.g., C₁-C₆alkylene), optionally substituted heteroalkylene (C₁-C₆ heteroalkylene),optionally substituted alkenylene (e.g., C₂-C₆ alkenylene), optionallysubstituted heteroalkenylene (e.g., C₂-C₆ heteroalkenylene), optionallysubstituted alkynylene (e.g., C₂-C₆ alkynylene), optionally substitutedheteroalkynylene (e.g., C₂-C₆ heteroalkynylene), optionally substitutedcycloalkylene, optionally substituted heterocycloalkylene, optionallysubstituted arylene, or optionally substituted heteroarylene;

Z is a chemical moiety formed from a coupling reaction between areactive substituent present on L and a reactive substituent presentwithin an antibody, antigen-binding fragment thereof, or ligand thatbinds CD45 (such as CD45RO), CD135, CD34, CD90, and/or CD110; and

wherein Am contains exactly one R_(C) substituent.

In some embodiments, R_(A) and R_(B), together with the oxygen atoms towhich they are bound, combine to form:

wherein Y is selected from O, S, NR_(E), and CR_(E)R_(E′), and

R_(E) and R_(E′) are each independently optionally substituted C₁-C₆alkylene-R_(C), optionally substituted C₁-C₆ heteroalkylene-R_(C),optionally substituted C₂-C₆ alkenylene-R_(C), optionally substitutedC₂-C₆ heteroalkenylene-R_(C), optionally substituted C₂-C₆alkynylene-R_(C), optionally substituted C₂-C₆ heteroalkynylene-R_(C),optionally substituted cycloalkylene-R_(C), optionally substitutedheterocycloalkylene-R_(C), optionally substituted arylene-R_(C), oroptionally substituted heteroarylene-R_(C).

In some embodiments, Am is represented by formula (IA) or formula (IB),

wherein R₁ is H, OH, OR_(A), or OR_(C);

R₂ is H, OH, OR_(B), or OR_(C);

R_(A) and R_(B), together with the oxygen atoms to which they are bound,combine to form:

R₃ is H or R_(C);

R₄ is H, OH, OR_(C), OR_(D), R_(C), or R_(D);

R₅ is H, OH, OR_(C), OR_(D), R_(C), or R_(D);

R₆ is H, OH, OR_(C), OR_(D), R_(C), or R_(D);

R₇ is H, OH, OR_(C), OR_(D), R_(C), or R_(D);

R₈ is OH, NH₂, OR_(C), or NHR_(C);

R₉ is H or OH; and

wherein R_(C) and R_(D) are each as defined above.

In some embodiments, Am is represented by formula (IA) or formula (IB),

wherein R₁ is H, OH, OR_(A), or OR_(C);

R₂ is H, OH, OR_(B), or OR_(C);

R_(A) and R_(B), together with the oxygen atoms to which they are bound,combine to form:

R₃ is H or R_(C);

R₄ and R₅ are each independently H, OH, OR_(C), R_(C), or OR_(D);

R₆ and R₇ are each H;

R₈ is OH, NH₂, OR_(C), or NHR_(C);

R₉ is H or OH; and

wherein R_(C) is as defined above.

In some embodiments, Am is represented by formula (IA) or formula (IB),

wherein R₁ is H, OH, or OR_(A);

R₂ is H, OH, or OR_(B);

R_(A) and R_(B), together with the oxygen atoms to which they are bound,combine to form:

R₃, R₄, R₆, and R₇ are each H;

R₅ is OR_(C);

R₈ is OH or NH₂;

R₉ is H or OH; and

wherein R_(C) is as defined above.

In some embodiments, Am is represented by formula (IA) or formula (IB),

wherein R₁ and R₂ are each independently H or OH;

R₃ is R_(C);

R₄, R₆, and R₇ are each H;

R₅ is H, OH, or OC₁-C₆ alkyl;

R₈ is OH or NH₂;

R₉ is H or OH; and

wherein R_(C) is as defined above.

In some embodiments, Am is represented by formula (IA) or formula (IB),

wherein R₁ and R₂ are each independently H or OH;

R₃, R₆, and R₇ are each H;

R₄ and R₅ are each independently H, OH, OR_(C), or R_(C);

R₈ is OH or NH₂;

R₉ is H or OH; and

wherein R_(C) is as defined above.

In some embodiments, Am is represented by formula (IA) or formula (IB),

wherein R₁ and R₂ are each independently H or OH;

R₃, R₆, and R₇ are each H;

R₄ and R₅ are each independently H or OH;

R₈ is OH, NH₂, OR_(C), or NHR_(C);

R₉ is H or OH; and

wherein R_(C) is as defined above.

In some embodiments, Am is represented by formula (II)

wherein X is S, SO, or SO₂; R₁ is H or a linker covalently bound to theantibody or antigen-binding fragment thereof; and R₂ is H or a linkercovalently bound to the antibody or antigen-binding fragment thereof;wherein when R₁ is H, R₂ is the linker, and when R₂ is H, R₁ is thelinker.

In some embodiments of any of the above aspects, the cytotoxin is amaytansinoid selected from the group consisting of DM1 and DM4. In someembodiments, the cytotoxin is an auristatin selected from the groupconsisting of monomethyl auristatin E and monomethyl auristatin F. Insome embodiments, the cytotoxin is an anthracycline selected from thegroup consisting of daunorubicin, doxorubicin, epirubicin, andidarubicin.

In some embodiments of any of the above aspects, the antibody orantigen-binding fragment thereof is selected from the group consistingof a monoclonal antibody or antigen-binding fragment thereof, apolyclonal antibody or antigen-binding fragment thereof, a humanizedantibody or antigen-binding fragment thereof, a bispecific antibody orantigen-binding fragment thereof, a dual-variable immunoglobulin domain,a single-chain Fv molecule (scFv), a diabody, a triabody, a nanobody, anantibody-like protein scaffold, a Fv fragment, a Fab fragment, a F(ab′)₂molecule, and a tandem di-scFv. In some embodiments, the antibody has anisotype selected from the group consisting of IgG, IgA, IgM, IgD, andIgE.

In some embodiments of any of the above aspects, the antibody,antigen-binding fragment thereof, or ligand is internalized by ahematopoietic cell, such as a hematopoietic stem cell, cancer cell, orautoimmune cell following administration to the patient. For instance,the antibody, antigen-binding fragment thereof, or ligand may beinternalized by hematopoietic stem cells, cancer cells, or autoimmunecells by receptor mediated endocytosis (e.g., upon binding tocell-surface CD45, CD135, CD34, CD90, or CD110). In some embodiments, acytotoxin covalently bound to the antibody or antigen-binding fragmentthereof may be released intracellularly by chemical cleavage (forinstance, by enzymatic or non-specific cleavage of a linker describedherein). The cytotoxin may then access its intracellular target (such asthe mitotic spindle apparatus, nuclear DNA, ribosomal RNA, ortopoisomerases, among others) so as to promote the death of anendogenous hematopoietic cell, such as an endogenous hematopoietic stemcell prior to transplantation therapy, an endogenous cancer cell, or anendogenous autoimmune cell, among others.

In some embodiments, of any of the above aspects, the antibody,antigen-binding fragment thereof, or ligand is capable of promotingnecrosis of a hematopoietic stem cell. In some embodiments, the antibodyor antigen-binding fragment thereof may promote the death of anendogenous hematopoietic stem cell prior to transplantation therapy byrecruiting one or more complement proteins, natural killer (NK) cells,macrophages, neutrophils, and/or eosinophils to the hematopoietic stemcell upon administration to the patient.

In some embodiments, the transplant containing hematopoietic stem cellsis administered to the patient after the concentration of the antibodyor antigen-binding fragment thereof has substantially cleared from theblood of the patient.

In some embodiments, the hematopoietic stem cells or progeny thereofmaintain hematopoietic stem cell functional potential after two or moredays (for example, from about 2 to about 5 days, from about 2 to about 7days, from about 2 to about 20 days, from about 2 to about 30 days, suchas 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26days, 27 days, 28 days, 29 days, 30 days, or more) followingtransplantation of the hematopoietic stem cells into the patient.

In some embodiments, the hematopoietic stem cells or progeny thereof arecapable of localizing to hematopoietic tissue, such as the bone marrow,and/or reestablishing hematopoiesis following transplantation of thehematopoietic stem cells into the patient.

In some embodiments, upon transplantation into the patient, thehematopoietic stem cells give rise to recovery of a population of cellsselected from the group consisting of megakaryocytes, thrombocytes,platelets, erythrocytes, mast cells, myeoblasts, basophils, neutrophils,eosinophils, microglia, granulocytes, monocytes, osteoclasts,antigen-presenting cells, macrophages, dendritic cells, natural killercells, T-lymphocytes, and B-lymphocytes.

In some embodiments of any of the above aspects, the method is used totreat one or more disorders, such as by depleting a population ofhematopoietic stem cells in a patient prior to hematopoietic stem celltransplant therapy so as to provide a niche to which the transplantedhematopoietic stem cells may home. Following transplantation, thehematopoietic stem cells may establish productive hematopoiesis, so asto replenish a deficient cell type in the patient or a cell type that isbeing actively killed or has been killed, for instance, bychemotherapeutic methods. For instance, the patient may be one that issuffering from a stem cell disorder. In some embodiments, the patient issuffering from a hemoglobinopathy disorder, such as sickle cell anemia,thalassemia, Fanconi anemia, aplastic anemia, and Wiskott-Aldrichsyndrome. The patient may be suffering from an immunodeficiencydisorder, such as a congenital immunodeficiency disorder or an acquiredimmunodeficiency disorder (e.g., human immunodeficiency virus oracquired immune deficiency syndrome). In some embodiments, the patientis suffering from a metabolic disorder, such as glycogen storagediseases, mucopolysaccharidoses, Gaucher's Disease, Hurlers Disease,sphingolipidoses, and metachromatic leukodystrophy. In some embodiments,the patient is suffering from a disorder selected from the groupconsisting of adenosine deaminase deficiency and severe combinedimmunodeficiency, hyper immunoglobulin M syndrome, Chediak-Higashidisease, hereditary lymphohistiocytosis, osteopetrosis, osteogenesisimperfecta, storage diseases, thalassemia major, systemic sclerosis,systemic lupus erythematosus, and juvenile rheumatoid arthritis. In someembodiments, the patient is suffering from an autoimmune disease, suchas scleroderma, multiple sclerosis, ulcerative colitis, Chron's disease,ant Type 1 diabetes. In some embodiments, the patient is suffering fromcancer or myeloproliferative disease, such as a hematological cancer. Insome embodiments, the patient is suffering from acute myeloid leukemia,acute lymphoid leukemia, chronic myeloid leukemia, chronic lymohoidleukemia, multiple meloma, diffuse large B-cell lymphoma, ornon-Hodgkin's lymphoma. In some embodiments, the patient is sufferingfrom a myelodysplastic disease, such as myelodysplastic syndrome.

In some embodiments of any of the above aspects, the method is used todirectly treat a cancer, such as a cancer characterized by CD45+,CD135+, CD34+, CD90+, or CD110+ cells (e.g., a leukemia characterized byCD45+, CD135+, CD34+, CD90+, or CD110+ cells), by administration of anantibody, antigen-binding fragment thereof, or ligand that depletes apopulation of CD45+, CD135+, CD34+, CD90+, or CD110+ cancer cells in thepatient and/or by administration of an antibody, antigen-bindingfragment thereof, or ligand so as to deplete a population of endogenoushematopoietic stem cells prior to hematopoietic stem celltransplantation. In the latter case, the transplantation may in turnre-constitute, for example, a population of cells depleted during theprocess of eradicating cancer cells. The cancer may be a hematologicalcancer, such as acute myeloid leukemia, acute lymphoid leukemia, chronicmyeloid leukemia, chronic lymohoid leukemia, multiple meloma, diffuselarge B-cell lymphoma, or non-Hodgkin's lymphoma.

In some embodiments of any of the above aspects, the method is used totreat an autoimmune disease, such as by administration of an antibody,antigen-binding fragment thereof, or ligand so as to deplete apopulation of CD45+, CD135+, CD34+, CD90+, or CD110+ autoimmune cellsand/or by administration of an antibody, antigen-binding fragmentthereof, or ligand so as to deplete a population of endogenoushematopoietic stem cells prior to hematopoietic stem celltransplantation. In the latter case, the transplantation may in turnre-constitute, for example, a population of cells depleted during theprocess of eradicating autoimmune cells. The autoimmune disease may be,for example, scleroderma, multiple sclerosis (MS), human systemic lupus(SLE), rheumatoid arthritis (RA), inflammatory bowel disease (IBD),treating psoriasis, Type 1 diabetes mellitus (Type 1 diabetes), acutedisseminated encephalomyelitis (ADEM), Addison's disease, alopeciauniversalis, ankylosing spondylitisis, antiphospholipid antibodysyndrome (APS), aplastic anemia, autoimmune hemolytic anemia, autoimmunehepatitis, autoimmune inner ear disease (AIED), autoimmunelymphoproliferative syndrome (ALPS), autoimmune oophoritis, Balodisease, Behcet's disease, bullous pemphigoid, cardiomyopathy, Chagas'disease, chronic fatigue immune dysfunction syndrome (CFIDS), chronicinflammatory demyelinating polyneuropathy, Crohn's disease, cicatricalpemphigoid, coeliac sprue-dermatitis herpetiformis, cold agglutinindisease, CREST syndrome, Degos disease, discoid lupus, dysautonomia,endometriosis, essential mixed cryoglobulinemia,fibromyalgia-fibromyositis, Goodpasture's syndrome, Grave's disease,Guillain-Barre syndrome (GBS), Hashimoto's thyroiditis, Hidradenitissuppurativa, idiopathic and/or acute thrombocytopenic purpura,idiopathic pulmonary fibrosis, IgA neuropathy, interstitial cystitis,juvenile arthritis, Kawasaki's disease, lichen planus, Lyme disease,Meniere disease, mixed connective tissue disease (MCTD), myastheniagravis, neuromyotonia, opsoclonus myoclonus syndrome (OMS), opticneuritis, Ord's thyroiditis, pemphigus vulgaris, pernicious anemia,polychondritis, polymyositis and dermatomyositis, primary biliarycirrhosis, polyarteritis nodosa, polyglandular syndromes, polymyalgiarheumatica, primary agammaglobulinemia, Raynaud phenomenon, Reiter'ssyndrome, rheumatic fever, sarcoidosis, scleroderma, Sjögren's syndrome,stiff person syndrome, Takayasu's arteritis, temporal arteritis (alsoknown as “giant cell arteritis”), ulcerative colitis, uveitis,vasculitis, vitiligo, vulvodynia (“vulvar vestibulitis”), and Wegener'sgranulomatosis.

Thus, in some embodiments of any of the above aspects, the inventionfeatures a method of treating a hemoglobinopathy disorder, such assickle cell anemia, thalassemia, Fanconi anemia, aplastic anemia, andWiskott-Aldrich syndrome. In some embodiments, the invention features amethod of treating an immunodeficiency disorder, such as a congenitalimmunodeficiency disorder or an acquired immunodeficiency disorder(e.g., human immunodeficiency virus or acquired immune deficiencysyndrome). In some embodiments, the invention features a method oftreating a metabolic disorder, such as glycogen storage diseases,mucopolysaccharidoses, Gaucher's Disease, Hurlers Disease,sphingolipidoses, and metachromatic leukodystrophy. In some embodiments,the invention features a method of treating a disorder selected from thegroup consisting of adenosine deaminase deficiency and severe combinedimmunodeficiency, hyper immunoglobulin M syndrome, Chediak-Higashidisease, hereditary lymphohistiocytosis, osteopetrosis, osteogenesisimperfecta, storage diseases, thalassemia major, systemic sclerosis,systemic lupus erythematosus, and juvenile rheumatoid arthritis In someembodiments, the invention features a method of treating an autoimmunedisease, such as scleroderma, multiple sclerosis, ulcerative colitis,Chron's disease, ant Type 1 diabetes. In some embodiments, the inventionfeatures a method of treating a cancer or myeloproliferative disease,such as a hematological cancer. In some embodiments, the inventionfeatures a method of treating acute myeloid leukemia, acute lymphoidleukemia, chronic myeloid leukemia, chronic lymohoid leukemia, multiplemeloma, diffuse large B-cell lymphoma, or non-Hodgkin's lymphoma. Insome embodiments, the patient is suffering from a myelodyplasticdisease, such as myelodysplastic syndrome. In these embodiments, themethod may include the steps of administering an antibody,antigen-binding fragment thereof, or ligand that binds CD45, CD135,CD34, CD90, or CD110 and/or a hematopoietic stem cell transplantaccording to the method of any of the above-described aspects andembodiments of the invention.

Similarly, in some embodiments of any of the above aspects, theinvention provides a method of treating cancer directly, such as acancer characterized by CD45+, CD135+, CD34+, CD90+, or CD110+ cells(e.g., a leukemia characterized by CD45+, CD135+, CD34+, CD90+, orCD110+ cells). In these embodiments, the method includes administeringan antibody, antigen-binding fragment thereof, or ligand that bindsCD45, CD135, CD34, CD90, or CD110. The cancer may be a hematologicalcancer, such as acute myeloid leukemia, acute lymphoid leukemia, chronicmyeloid leukemia, chronic lymohoid leukemia, multiple meloma, diffuselarge B-cell lymphoma, or non-Hodgkin's lymphoma.

Additionally, in some embodiments of any of the above aspects, theinvention provides a method of treating an autoimmune disease, such asmultiple sclerosis (MS), human systemic lupus (SLE), rheumatoidarthritis (RA), inflammatory bowel disease (IBD), treating psoriasis,Type 1 diabetes mellitus (Type 1 diabetes) acute disseminatedencephalomyelitis (ADEM), Addison's disease, alopecia universalis,ankylosing spondylitisis, antiphospholipid antibody syndrome (APS),aplastic anemia, autoimmune hemolytic anemia, autoimmune hepatitis,autoimmune inner ear disease (AIED), autoimmune lymphoproliferativesyndrome (ALPS), autoimmune oophoritis, Balo disease, Behcet's disease,bullous pemphigoid, cardiomyopathy, Chagas' disease, chronic fatigueimmune dysfunction syndrome (CFIDS), chronic inflammatory demyelinatingpolyneuropathy, Crohn's disease, cicatrical pemphigoid, coeliacsprue-dermatitis herpetiformis, cold agglutinin disease, CREST syndrome,Degos disease, discoid lupus, dysautonomia, endometriosis, essentialmixed cryoglobulinemia, fibromyalgia-fibromyositis, Goodpasture'ssyndrome, Grave's disease, Guillain-Barre syndrome (GBS), Hashimoto'sthyroiditis, Hidradenitis suppurativa, idiopathic and/or acutethrombocytopenic purpura, idiopathic pulmonary fibrosis, IgA neuropathy,interstitial cystitis, juvenile arthritis, Kawasaki's disease, lichenplanus, Lyme disease, Meniere disease, mixed connective tissue disease(MCTD), myasthenia gravis, neuromyotonia, opsoclonus myoclonus syndrome(OMS), optic neuritis, Ord's thyroiditis, pemphigus vulgaris, perniciousanemia, polychondritis, polymyositis and dermatomyositis, primarybiliary cirrhosis, polyarteritis nodosa, polyglandular syndromes,polymyalgia rheumatica, primary agammaglobulinemia, Raynaud phenomenon,Reiter's syndrome, rheumatic fever, sarcoidosis, scleroderma, Sjögren'ssyndrome, stiff person syndrome, Takayasu's arteritis, temporalarteritis (also known as “giant cell arteritis”), ulcerative colitis,uveitis, vasculitis, vitiligo, vulvodynia (“vulvar vestibulitis”), andWegener's granulomatosis. In these embodiments, the method includesadministering an antibody, antigen-binding fragment thereof, or ligandthat binds CD45, CD135, CD34, CD90, or CD110.

In another aspect, the invention features a method of depleting apopulation of CD45+ cells by contacting the population with an effectiveamount of a conjugate represented by the formula Ab-Am, wherein Ab is anantibody or antigen-binding fragment thereof that binds CD45 and Am isan amatoxin. Am may be represented by formula (IA)

wherein R₁ is H, OH, OR_(A), or OR_(C);

R₂ is H, OH, OR_(B), or OR_(C);

R_(A) and R_(B), together with the oxygen atoms to which they are bound,combine to form an optionally substituted 5-membered heterocyclolalkylgroup;

R₃ is H, R_(C), or R_(D);

R₄, R₅, R₆, and R₇ are each independently H, OH, OR_(C), OR_(D), R_(C),or R_(D);

R₈ is OH, NH₂, OR_(C), OR_(D), NHR_(C), or NR_(C)R_(D);

R₉ is H, OH, OR_(C), or OR_(D);

X is —S—, —S(O)—, or —SO₂—;

R_(C) is -L-Z;

R_(D) is optionally substituted alkyl (e.g., C₁-C₆ alkyl), optionallysubstituted heteroalkyl (e.g., C₁-C₆ heteroalkyl), optionallysubstituted alkenyl (e.g., C₂-C₆ alkenyl), optionally substitutedheteroalkenyl (e.g., C₂-C₆ heteroalkenyl), optionally substitutedalkynyl (e.g., C₂-C₆ alkynyl), optionally substituted heteroalkynyl(e.g., C₂-C₆ heteroalkynyl), optionally substituted cycloalkyl,optionally substituted heterocycloalkyl, optionally substituted aryl, oroptionally substituted heteroaryl;

L is a linker, such as optionally substituted alkylene (e.g., C₁-C₆alkylene), optionally substituted heteroalkylene (C₁-C₆ heteroalkylene),optionally substituted alkenylene (e.g., C₂-C₆ alkenylene), optionallysubstituted heteroalkenylene (e.g., C₂-C₆ heteroalkenylene), optionallysubstituted alkynylene (e.g., C₂-C₆ alkynylene), optionally substitutedheteroalkynylene (e.g., C₂-C₆ heteroalkynylene), optionally substitutedcycloalkylene, optionally substituted heterocycloalkylene, optionallysubstituted arylene, or optionally substituted heteroarylene; and

Z is a chemical moiety formed from a coupling reaction between areactive substituent present on L and a reactive substituent presentwithin the antibody or antigen-binding fragment thereof,

wherein Am contains exactly one R_(C) substituent.

In some embodiments, Am is represented by formula (IB)

wherein R₁ is H, OH, OR_(A), or OR_(C);

R₂ is H, OH, OR_(B), or OR_(C);

R_(A) and R_(B), together with the oxygen atoms to which they are bound,combine to form an optionally substituted 5-membered heterocyclolalkylgroup;

R₃ is H, R_(C), or R_(D);

R₄, R₅, R₆, and R₇ are each independently H, OH, OR_(C), OR_(D), R_(C),or R_(D);

R₈ is OH, NH₂, OR_(C), OR_(D), NHR_(C), or NR_(C)R_(D);

R₉ is H, OH, OR_(C), or OR_(D);

X is —S—, —S(O)—, or —SO₂—;

R_(C) is -L-Z;

R_(D) is optionally substituted alkyl (e.g., C₁-C₆ alkyl), optionallysubstituted heteroalkyl (e.g., C₁-C₆ heteroalkyl), optionallysubstituted alkenyl (e.g., C₂-C₆ alkenyl), optionally substitutedheteroalkenyl (e.g., C₂-C₆ heteroalkenyl), optionally substitutedalkynyl (e.g., C₂-C₆ alkynyl), optionally substituted heteroalkynyl(e.g., C₂-C₆ heteroalkynyl), optionally substituted cycloalkyl,optionally substituted heterocycloalkyl, optionally substituted aryl, oroptionally substituted heteroaryl;

L is a linker, such as optionally substituted alkylene (e.g., C₁-C₆alkylene), optionally substituted heteroalkylene (C₁-C₆ heteroalkylene),optionally substituted alkenylene (e.g., C₂-C₆ alkenylene), optionallysubstituted heteroalkenylene (e.g., C₂-C₆ heteroalkenylene), optionallysubstituted alkynylene (e.g., C₂-C₆ alkynylene), optionally substitutedheteroalkynylene (e.g., C₂-C₆ heteroalkynylene), optionally substitutedcycloalkylene, optionally substituted heterocycloalkylene, optionallysubstituted arylene, or optionally substituted heteroarylene; and

Z is a chemical moiety formed from a coupling reaction between areactive substituent present on L and a reactive substituent presentwithin the antibody or antigen-binding fragment thereof,

wherein Am contains exactly one R_(C) substituent.

In another aspect, the invention features a conjugate represented by theformula Ab-Am, wherein Ab is an antibody or antigen-binding fragmentthereof that binds CD45 and Am is an amatoxin. In some embodiments, Amis represented by formula (IA) or formula (IB), above.

In some embodiments of the preceding two aspects, the antibody orantigen-binding fragment thereof is conjugated to the amatoxin by way ofa cysteine residue in the Fc domain of the antibody or antigen-bindingfragment thereof. In some embodiments, the cysteine residue isintroduced by way of a mutation in the Fc domain of the antibody orantigen-binding fragment thereof. For instance, the cysteine residue maybe selected from the group consisting of Cys118, Cys239, and Cys265.

In some embodiments of these aspects, the cysteine residue is naturallyoccurring in the Fc domain of the antibody or antigen-binding fragmentthereof. For instance, the Fc domain may be an IgG Fc domain, such as ahuman IgG1 Fc domain, and the cysteine residue may be selected from thegroup consisting of Cys261, Csy321, Cys367, and Cys425.

In some embodiments of these aspects, R₁ is H, OH, or OR_(A);

R₂ is H, OH, or OR_(B);

R_(A) and R_(B), together with the oxygen atoms to which they are bound,combine to form:

R₃, R₄, R₆, and R₇ are each H;

R₅ is OR_(C);

R₈ is OH or NH₂; and

R₉ is H or OH.

In some embodiments, R₁ and R₂ are each independently H or OH;

R₃ is R_(C);

R₄, R₆, and R₇ are each H;

R₅ is H, OH, or OC₁-C₆ alkyl;

R₈ is OH or NH₂; and

R₉ is H or OH.

In some embodiments, R₁ and R₂ are each independently H or OH;

R₃, R₆, and R₇ are each H;

R₄ is OR_(C), or R_(C);

R₅ is H, OH, or OC₁-C₆ alkyl;

R₈ is OH or NH₂; and

R₉ is H or OH.

In some embodiments, R₁ and R₂ are each independently H or OH;

R₃, R₆, and R₇ are each H;

R₄ and R₅ are each independently H or OH;

R₈ is OR_(C) or NHR_(C); and

R₉ is H or OH.

In some embodiments of these aspects, the antibody or antigen-bindingfragment thereof is internalized by a CD45+ cell.

In some embodiments of these aspects, the antibody or antigen-bindingfragment thereof binds CD45 with a K_(d) of less than 1 μM, less than750 nM, less than 500 nM, less than 250 nM, less than 200 nM, less than150 nM, less than 100 nM, less than 75 nM, less than 50 nM, less than 10nM, less than 1 nM, less than 0.1 nM, less than 10 pM, less than 1 pM,or less than 0.1 pM. In some embodiments, the K_(d) is from about 0.1 pMto about 1 μM. In some embodiments, the K_(d) is about 357 pM, thek_(on) is about 2.56×10⁵ M⁻¹s⁻¹, and/or the k_(off) is about 9.14×10⁻⁵s⁻¹.

In some embodiments of these aspects, the antibody or antigen-bindingfragment thereof binds CD45 with a k_(on) of from about 9×10⁻² M⁻¹ s⁻¹to about 1×10² M⁻¹s⁻¹.

In some embodiments of these aspects, the antibody or antigen-bindingfragment thereof competitively inhibits the binding of CD45 to a secondantibody or antigen binding fragment thereof, wherein the secondantibody or antigen-binding fragment thereof has the followingcomplementarity determining regions (CDRs):

a. a CDR-H1 having the amino acid sequence SYAMS; (SEQ ID NO: 16)b. a CDR-H2 having the amino acid sequence AISGSGGSTFYADSVRG;(SEQ ID NO: 17) c. a CDR-H3 having the amino acid sequence EVMGPIFFDY;(SEQ ID NO: 18) d. a CDR-L1 having the amino acid sequence RASQSIISSALA;(SEQ ID NO: 19) e. a CDR-L2 having the amino acid sequence GASSRAT;(SEQ ID NO: 20) and f. a CDR-L3 having the amino acid sequenceQQYGSTPLT. (SEQ ID NO: 21)

In some embodiments of these aspects, the antibody or antigen-bindingfragment thereof is rat YTH24.5 or a humanized variant thereof.

In some embodiments of these aspects, the antibody or antigen-bindingfragment thereof competitively inhibits the binding of CD45 to a secondantibody or antigen binding fragment thereof, wherein the secondantibody or antigen-binding fragment thereof is rat YTH24.5 or ahumanized variant thereof.

In some embodiments of these aspects, the antibody or antigen-bindingfragment thereof is selected from the group consisting of a monoclonalantibody or antigen-binding fragment thereof, a polyclonal antibody orantigen-binding fragment thereof, a humanized antibody orantigen-binding fragment thereof, a bispecific antibody orantigen-binding fragment thereof, a dual-variable immunoglobulin domain,a single-chain Fv molecule (scFv), a diabody, a triabody, a nanobody, anantibody-like protein scaffold, a Fv fragment, a Fab fragment, a F(ab′)₂molecule, and a tandem di-scFV.

In another aspect, the invention features a method of depleting apopulation of CD135+ cells by contacting the population with aneffective amount of a conjugate represented by the formula Ab-Am,wherein Ab is an antibody or antigen-binding fragment thereof that bindsCD135 and Am is an amatoxin. Am may be represented by formula (IA)

wherein R₁ is H, OH, OR_(A), or OR_(C);

R₂ is H, OH, OR_(B), or OR_(C);

R_(A) and R_(B), together with the oxygen atoms to which they are bound,combine to form an optionally substituted 5-membered heterocyclolalkylgroup;

R₃ is H, R_(C), or R_(D);

R₄, R₅, R₆, and R₇ are each independently H, OH, OR_(C), OR_(D), R_(C),or R_(D);

R₈ is OH, NH₂, OR_(C), OR_(D), NHR_(C), or NR_(C)R_(D);

R₉ is H, OH, OR_(C), or OR_(D);

X is —S—, —S(O)—, or —SO₂—;

R_(C) is -L-Z;

R_(D) is optionally substituted alkyl (e.g., C₁-C₆ alkyl), optionallysubstituted heteroalkyl (e.g., C₁-C₆ heteroalkyl), optionallysubstituted alkenyl (e.g., C₂-C₆ alkenyl), optionally substitutedheteroalkenyl (e.g., C₂-C₆ heteroalkenyl), optionally substitutedalkynyl (e.g., C₂-C₆ alkynyl), optionally substituted heteroalkynyl(e.g., C₂-C₆ heteroalkynyl), optionally substituted cycloalkyl,optionally substituted heterocycloalkyl, optionally substituted aryl, oroptionally substituted heteroaryl;

L is a linker, such as optionally substituted alkylene (e.g., C₁-C₆alkylene), optionally substituted heteroalkylene (C₁-C₆ heteroalkylene),optionally substituted alkenylene (e.g., C₂-C₆ alkenylene), optionallysubstituted heteroalkenylene (e.g., C₂-C₆ heteroalkenylene), optionallysubstituted alkynylene (e.g., C₂-C₆ alkynylene), optionally substitutedheteroalkynylene (e.g., C₂-C₆ heteroalkynylene), optionally substitutedcycloalkylene, optionally substituted heterocycloalkylene, optionallysubstituted arylene, or optionally substituted heteroarylene; and

Z is a chemical moiety formed from a coupling reaction between areactive substituent present on L and a reactive substituent presentwithin the antibody or antigen-binding fragment thereof,

wherein Am contains exactly one R_(C) substituent.

In some embodiments, Am is represented by formula (IB)

wherein R₁ is H, OH, OR_(A), or OR_(C);

R₂ is H, OH, OR_(B), or OR_(C);

R_(A) and R_(B), together with the oxygen atoms to which they are bound,combine to form an optionally substituted 5-membered heterocyclolalkylgroup;

R₃ is H, R_(C), or R_(D);

R₄, R₅, R₆, and R₇ are each independently H, OH, OR_(C), OR_(D), R_(C),or R_(D);

R₈ is OH, NH₂, OR_(C), OR_(D), NHR_(C), or NR_(C)R_(D);

R₉ is H, OH, OR_(C), or OR_(D);

X is —S—, —S(O)—, or —SO₂—;

R_(C) is -L-Z;

R_(D) is optionally substituted alkyl (e.g., C₁-C₆ alkyl), optionallysubstituted heteroalkyl (e.g., C₁-C₆ heteroalkyl), optionallysubstituted alkenyl (e.g., C₂-C₆ alkenyl), optionally substitutedheteroalkenyl (e.g., C₂-C₆ heteroalkenyl), optionally substitutedalkynyl (e.g., C₂-C₆ alkynyl), optionally substituted heteroalkynyl(e.g., C₂-C₆ heteroalkynyl), optionally substituted cycloalkyl,optionally substituted heterocycloalkyl, optionally substituted aryl, oroptionally substituted heteroaryl;

L is a linker, such as optionally substituted alkylene (e.g., C₁-C₆alkylene), optionally substituted heteroalkylene (C₁-C₆ heteroalkylene),optionally substituted alkenylene (e.g., C₂-C₆ alkenylene), optionallysubstituted heteroalkenylene (e.g., C₂-C₆ heteroalkenylene), optionallysubstituted alkynylene (e.g., C₂-C₆ alkynylene), optionally substitutedheteroalkynylene (e.g., C₂-C₆ heteroalkynylene), optionally substitutedcycloalkylene, optionally substituted heterocycloalkylene, optionallysubstituted arylene, or optionally substituted heteroarylene; and

Z is a chemical moiety formed from a coupling reaction between areactive substituent present on L and a reactive substituent presentwithin the antibody or antigen-binding fragment thereof,

wherein Am contains exactly one R_(C) substituent.

In another aspect, the invention features a conjugate represented by theformula Ab-Am, wherein Ab is an antibody or antigen-binding fragmentthereof that binds CD135 and Am is an amatoxin. In some embodiments, Amis represented by formula (IA) or formula (IB), above.

In some embodiments of the preceding two aspects, the antibody orantigen-binding fragment thereof is conjugated to the amatoxin by way ofa cysteine residue in the Fc domain of the antibody or antigen-bindingfragment thereof. In some embodiments, the cysteine residue isintroduced by way of a mutation in the Fc domain of the antibody orantigen-binding fragment thereof. For instance, the cysteine residue maybe selected from the group consisting of Cys118, Cys239, and Cys265.

In some embodiments of these aspects, the cysteine residue is naturallyoccurring in the Fc domain of the antibody or antigen-binding fragmentthereof. For instance, the Fc domain may be an IgG Fc domain, such as ahuman IgG1 Fc domain, and the cysteine residue may be selected from thegroup consisting of Cys261, Csy321, Cys367, and Cys425.

In some embodiments of these aspects, R₁ is H, OH, or OR_(A);

R₂ is H, OH, or OR_(B);

R_(A) and R_(B), together with the oxygen atoms to which they are bound,combine to form:

R₃, R₄, R₆, and R₇ are each H;

R₅ is OR_(C);

R₈ is OH or NH₂; and

R₉ is H or OH.

In some embodiments, R₁ and R₂ are each independently H or OH;

R₃ is R_(C);

R₄, R₆, and R₇ are each H;

R₅ is H, OH, or OC₁-C₆ alkyl;

R₈ is OH or NH₂; and

R₉ is H or OH.

In some embodiments, R₁ and R₂ are each independently H or OH;

R₃, R₆, and R₇ are each H;

R₄ is OR_(C), or R_(C);

R₅ is H, OH, or OC₁-C₆ alkyl;

R₈ is OH or NH₂; and

R₉ is H or OH.

In some embodiments, R₁ and R₂ are each independently H or OH;

R₃, R₆, and R₇ are each H;

R₄ and R₅ are each independently H or OH;

R₈ is OR_(C) or NHR_(C); and

R₉ is H or OH.

In some embodiments of these aspects, the antibody or antigen-bindingfragment thereof is internalized by a CD135+ cell.

In some embodiments of these aspects, the antibody or antigen-bindingfragment thereof binds CD135 with a K_(d) of less than 1 μM, less than750 nM, less than 500 nM, less than 250 nM, less than 200 nM, less than150 nM, less than 100 nM, less than 75 nM, less than 50 nM, less than 10nM, less than 1 nM, less than 0.1 nM, less than 10 pM, less than 1 pM,or less than 0.1 pM, In some embodiments, the K_(d) is from about 0.1 pMto about 1 μM.

In some embodiments of these aspects, the antibody or antigen-bindingfragment thereof binds CD135 with a k_(on) of from about 9×10⁻² M⁻¹ s⁻¹to about 1×10² M⁻¹s⁻¹.

In some embodiments of these aspects, the antibody or antigen-bindingfragment thereof has the following CDRs:

a. a CDR-H1 having the amino acid sequence SYYMH; (SEQ ID NO: 1)b. a CDR-H2 having the amino acid sequence IINPSGGSTSYAQKFQG;(SEQ ID NO: 2) c. a CDR-H3 having the amino acid sequence GVGAHDAFDI(SEQ ID NO: 3) or VVAAAVADY; (SEQ ID NO: 4)d. a CDR-L1 having the amino acid sequence RSSQSLLHSNGNNYLD(SEQ ID NO: 5) or RSSQSLLHSNGYNYLD; (SEQ ID NO: 6)e. a CDR-L2 having the amino acid sequence LGSNRAS; (SEQ ID NO: 7) andf. a CDR-L3 having the amino acid sequence MQGTHPAIS (SEQ ID NO: 8) orMQSLQTPFT. (SEQ ID NO: 9)

In some embodiments of these aspects, the antibody or antigen-bindingfragment thereof has the following CDRs:

a. a CDR-H1 having the amino acid sequence SYAIS; (SEQ ID NO: 10)b. a CDR-H2 having the amino acid sequence GIIPIFGTANYAQKFQG;(SEQ ID NO: 11) c. a CDR-H3 having the amino acid sequenceFALFGFREQAFDI; (SEQ ID NO: 12)d. a CDR-L1 having the amino acid sequence RASQSISSYLN; (SEQ ID NO: 13)e. a CDR-L2 having the amino acid sequence AASSLQS; (SEQ ID NO: 14) andf. a CDR-L3 having the amino acid sequence QQSYSTPFT. (SEQ ID NO: 15)

In some embodiments of these aspects, the antibody or antigen-bindingfragment thereof competitively inhibits the binding of CD135 to a secondantibody or antigen binding fragment thereof, wherein the secondantibody or antigen-binding fragment thereof has the following CDRs:

a. a CDR-H1 having the amino acid sequence SYYMH; (SEQ ID NO: 1)b. a CDR-H2 having the amino acid sequence IINPSGGSTSYAQKFQG;(SEQ ID NO: 2) c. a CDR-H3 having the amino acid sequence GVGAHDAFDI(SEQ ID NO: 3) or VVAAAVADY; (SEQ ID NO: 4)d. a CDR-L1 having the amino acid sequence RSSQSLLHSNGNNYLD(SEQ ID NO: 5) or RSSQSLLHSNGYNYLD; (SEQ ID NO: 6)e. a CDR-L2 having the amino acid sequence LGSNRAS; (SEQ ID NO: 7) andf. a CDR-L3 having the amino acid sequence MQGTHPAIS (SEQ ID NO: 8) orMQSLQTPFT. (SEQ ID NO: 9)

In some embodiments of these aspects, the antibody or antigen-bindingfragment thereof competitively inhibits the binding of CD135 to a secondantibody or antigen binding fragment thereof, wherein the secondantibody or antigen-binding fragment thereof has the following CDRs:

(SEQ ID NO: 10) a. a CDR-H1 having the amino acid sequence SYAIS;(SEQ ID NO: 11) b. a CDR-H2 having the amino acid sequenceGIIPIFGTANYAQKFQG; (SEQ ID NO: 12)c. a CDR-H3 having the amino acid sequence FALFGFREQAFDI;(SEQ ID NO: 13) d. a CDR-L1 having the amino acid sequence RASQSISSYLN;(SEQ ID NO: 14) e. a CDR-L2 having the amino acid sequence AASSLQS; and(SEQ ID NO: 15) f. a CDR-L3 having the amino acid sequence QQSYSTPFT.

In some embodiments of these aspects, the antibody or antigen-bindingfragment thereof is selected from the group consisting of a monoclonalantibody or antigen-binding fragment thereof, a polyclonal antibody orantigen-binding fragment thereof, a humanized antibody orantigen-binding fragment thereof, a bispecific antibody orantigen-binding fragment thereof, a dual-variable immunoglobulin domain,a single-chain Fv molecule (scFv), a diabody, a triabody, a nanobody, anantibody-like protein scaffold, a Fv fragment, a Fab fragment, a F(ab′)₂molecule, and a tandem di-scFV.

In another aspect, the invention features a conjugate represented by theformula Ab-Cy, wherein Ab is an antibody or antigen-binding fragmentthereof that binds CD45 (e.g., CD45RO) and Cy is a cytotoxin. In someembodiments of this aspect, the cytotoxin is pseudomonas exotoxin A,deBouganin, diphtheria toxin, saporin, maytansine, a maytansinoid, anauristatin, an anthracycline, a calicheamicin, irinotecan, SN-38, aduocarmycin, a pyrrolobenzodiazepine, a pyrrolobenzodiazepine dimer, anindolinobenzodiazepine, or an indolinobenzodiazepine dimer, or a variantof any of the foregoing cytotoxins.

In another aspect, the invention features a conjugate represented by theformula Ab-Cy, wherein Ab is an antibody or antigen-binding fragmentthereof that binds CD135 and Cy is a cytotoxin. In some embodiments ofthis aspect, the cytotoxin is pseudomonas exotoxin A, deBouganin,diphtheria toxin, saporin, maytansine, a maytansinoid, an auristatin, ananthracycline, a calicheamicin, irinotecan, SN-38, a duocarmycin, apyrrolobenzodiazepine, a pyrrolobenzodiazepine dimer, anindolinobenzodiazepine, or an indolinobenzodiazepine dimer, or a variantof any of the foregoing cytotoxins.

In another aspect, the invention features a conjugate represented by theformula Ab-Cy, wherein Ab is an antibody or antigen-binding fragmentthereof that binds CD34 and Cy is a cytotoxin. In some embodiments ofthis aspect, the cytotoxin is pseudomonas exotoxin A, deBouganin,diphtheria toxin, saporin, maytansine, a maytansinoid, an auristatin, ananthracycline, a calicheamicin, irinotecan, SN-38, a duocarmycin, apyrrolobenzodiazepine, a pyrrolobenzodiazepine dimer, anindolinobenzodiazepine, or an indolinobenzodiazepine dimer, or a variantof any of the foregoing cytotoxins.

In another aspect, the invention features a conjugate represented by theformula Ab-Cy, wherein Ab is an antibody or antigen-binding fragmentthereof that binds CD90 and Cy is a cytotoxin. In some embodiments ofthis aspect, the cytotoxin is pseudomonas exotoxin A, deBouganin,diphtheria toxin, saporin, maytansine, a maytansinoid, an auristatin, ananthracycline, a calicheamicin, irinotecan, SN-38, a duocarmycin, apyrrolobenzodiazepine, a pyrrolobenzodiazepine dimer, anindolinobenzodiazepine, or an indolinobenzodiazepine dimer, or a variantof any of the foregoing cytotoxins.

In another aspect, the invention features a conjugate represented by theformula Ab-Cy, wherein Ab is an antibody or antigen-binding fragmentthereof that binds CD110 and Cy is a cytotoxin. In some embodiments ofthis aspect, the cytotoxin is pseudomonas exotoxin A, deBouganin,diphtheria toxin, saporin, maytansine, a maytansinoid, an auristatin, ananthracycline, a calicheamicin, irinotecan, SN-38, a duocarmycin, apyrrolobenzodiazepine, a pyrrolobenzodiazepine dimer, anindolinobenzodiazepine, or an indolinobenzodiazepine dimer, or a variantof any of the foregoing cytotoxins.

In some embodiments, of the foregoing five aspects, the antibody orantigen-binding fragment thereof is internalized by a hematopoieticcell, such as a hematopoietic stem cell (e.g., a CD45+(such as aCD45RO+), CD135+, CD34+, CD90+, and/or CD110+ cell).

In some embodiments of these aspects, the antibody or antigen-bindingfragment thereof binds CD45 (e.g., CD45RO), CD135, CD34, CD90, or CD110with a K_(d) of less than 1 μM, less than 750 nM, less than 500 nM, lessthan 250 nM, less than 200 nM, less than 150 nM, less than 100 nM, lessthan 75 nM, less than 50 nM, less than 10 nM, less than 1 nM, less than0.1 nM, less than 10 pM, less than 1 pM, or less than 0.1 pM. In someembodiments, the K_(d) is from about 0.1 pM to about 1 μM.

In some embodiments of this aspect, the antibody or antigen-bindingfragment thereof binds CD45 (e.g., CD45RO), CD135, CD34, CD90, or CD110with a k_(on) of from about 9×10⁻² M⁻¹ s⁻¹ to about 1×10² M⁻¹s⁻¹.

In some embodiments of these aspects, the antibody or antigen-bindingfragment thereof is selected from the group consisting of a monoclonalantibody or antigen-binding fragment thereof, a polyclonal antibody orantigen-binding fragment thereof, a humanized antibody orantigen-binding fragment thereof, a bispecific antibody orantigen-binding fragment thereof, a dual-variable immunoglobulin domain,a single-chain Fv molecule (scFv), a diabody, a triabody, a nanobody, anantibody-like protein scaffold, a Fv fragment, a Fab fragment, a F(ab′)₂molecule, and a tandem di-scFv. In some embodiments, the antibody has anisotype selected from the group consisting of IgG, IgA, IgM, IgD, andIgE.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph demonstrating the effect of various concentrations ofanti-CD45 monoclonal antibodies Ab1, Ab2, and Ab3, or isotype-matchednegative controls, each bound to saporin by way of saporin-conjugatedFab fragments, on the viability of Reh cells in vitro. Cell viabilitywas assessed using the CellTiter-Glo™ assay kit as described in Example8, below.

FIG. 2 is a graph demonstrating the effect of various concentrations ofanti-CD45 monoclonal antibody or isotype-matched negative control, eachbound to saporin by way of saporin-conjugated Fab fragments, on theviability of human CD34+ cells in vitro. Cell viability was assessedusing the CellTiter-Glo™ assay kit as described in Example 8, below.

DETAILED DESCRIPTION

The invention provides methods of treating a variety of disorders, suchas diseases of a cell type in the hematopoietic lineage, cancers,autoimmune diseases, metabolic disorders, and stem cell disorders, amongothers. The compositions and methods described herein may (i) directlydeplete a population of cells that give rise to a pathology, such as apopulation of cancer cells (e.g., leukemia cells) and autoimmune cells(e.g., autoreactive T-cells), and/or (ii) deplete a population ofendogenous hematopoietic stem cells so as to promote the engraftment oftransplanted hematopoietic stem cells by providing a niche to which thetransplanted cells may home. The foregoing activities can be achieved byadministration of an antibody, antigen-binding fragment thereof, orligand capable of binding an antigen expressed by an endogenousdisease-causing cell or a hematopoietic stem cell. In the case of directtreatment of a disease, this administration can cause a reduction in thequantity of the cells that give rise to the pathology of interest. Inthe case of preparing a patient for hematopoietic stem cell transplanttherapy, this administration can cause the selective depletion of apopulation of endogenous hematopoietic stem cells, thereby creating avacancy in the hematopoietic tissue, such as the bone marrow, that cansubsequently be filled by transplanted, exogenous hematopoietic stemcells. The invention is based in part on the discovery that antibodies,antigen-binding fragments thereof, and ligands capable of binding CD45,CD135, CD34, CD90, or CD110 can be administered to a patient to effectboth of the above activities. Antibodies, antigen-binding fragmentsthereof, and ligands that bind CD45, CD135, CD34, CD90, or CD110 can beadministered to a patient suffering from a cancer or autoimmune diseaseto directly deplete a population of cancerous cells or autoimmune cells,and can also be administered to a patient in need of hematopoietic stemcell transplant therapy in order to promote the survival and engraftmentpotential of transplanted hematopoietic stem cells.

Engraftment of hematopoietic stem cell transplants due to theadministration of anti-CD45, anti-CD135, anti-CD34, anti-CD90, oranti-CD110 antibodies, antigen-binding fragments thereof, or ligands canmanifest in a variety of empirical measurements. For instance,engraftment of transplanted hematopoietic stem cells can be evaluated byassessing the quantity of competitive repopulating units (CRU) presentwithin the bone marrow of a patient following administration of anantibody or antigen-binding fragment thereof capable of binding CD45,CD135, CD34, CD90, or CD110 and subsequent administration of ahematopoietic stem cell transplant. Additionally, one can observeengraftment of a hematopoietic stem cell transplant by incorporating areporter gene, such as an enzyme that catalyzes a chemical reactionyielding a fluorescent, chromophoric, or luminescent product, into avector with which the donor hematopoietic stem cells have beentransfected and subsequently monitoring the corresponding signal in atissue into which the hematopoietic stem cells have homed, such as thebone marrow. One can also observe hematopoietic stem cell engraftment byevaluation of the quantity and survival of hematopoietic stem andprogenitor cells, for instance, as determined by fluorescence activatedcell sorting (FACS) analysis methods known in the art. Engraftment canalso be determined by measuring white blood cell counts in peripheralblood during a post-transplant period, and/or by measuring recovery ofmarrow cells by donor cells in a bone marrow aspirate sample.

The sections that follow provide a description of antibodies,antigen-binding fragments thereof, and ligands that can be administeredto a patient, such as a patient suffering from a cancer or autoimmunedisease, or a patient in need of hematopoietic stem cell transplanttherapy in order to promote engraftment of hematopoietic stem cellgrafts, as well as methods of administering such therapeutics to apatient (e.g., prior to hematopoietic stem cell transplantation).

Definitions

As used herein, the term “about” refers to a value that is within 10%above or below the value being described. For example, the term “about 5nM” indicates a range of from 4.5 nM to 5.5 nM.

As used herein, the term “amatoxin” refers to a member of the amatoxinfamily of peptides produced by Amanita phalloides mushrooms, or avariant or derivative thereof, such as a variant or derivative thereofcapable of inhibiting RNA polymerase II activity. Amatoxins useful inconjunction with the compositions and methods described herein includecompounds according to formula (III), below, such as α-amanitin,β-amanitin, γ-amanitin, ε-amanitin, amanin, amaninamide, amanullin,amanullinic acid, and proamanullin. Formula (III) is as follows:

wherein R₁ is H, OH, or OR_(A);

R₂ is H, OH, or OR_(B);

R_(A) and R_(B), together with the oxygen atoms to which they are bound,combine to form an optionally substituted 5-membered heterocyclolalkylgroup;

R₃ is H or R_(D);

R₄ is H, OH, OR_(D), or R_(D);

R₅ is H, OH, OR_(D), or R_(D);

R₆ is H, OH, OR_(D), or R_(D);

R₇ is H, OH, OR_(D), or R_(D);

R₈ is OH, NH₂, or OR_(D);

R₉ is H, OH, or OR_(D);

X is —S—, —S(O)—, or —SO₂—; and

R_(D) is optionally substituted alkyl (e.g., C₁-C₆ alkyl), optionallysubstituted heteroalkyl (e.g., C₁-C₆ heteroalkyl), optionallysubstituted alkenyl (e.g., C₂-C₆ alkenyl), optionally substitutedheteroalkenyl (e.g., C₂-C₆ heteroalkenyl), optionally substitutedalkynyl (e.g., C₂-C₆ alkynyl), optionally substituted heteroalkynyl(e.g., C₂-C₆ heteroalkynyl), optionally substituted cycloalkyl,optionally substituted heterocycloalkyl, optionally substituted aryl, oroptionally substituted heteroaryl.

For instance, amatoxins useful in conjunction with the compositions andmethods described herein include compounds according to formula (IIIA),below:

wherein R₁ is H, OH, or OR_(A);

R₂ is H, OH, or OR_(B);

R_(A) and R_(B), together with the oxygen atoms to which they are bound,combine to form an optionally substituted 5-membered heterocyclolalkylgroup;

R₃ is H or R_(D);

R₄ is H, OH, OR_(D), or R_(D);

R₅ is H, OH, OR_(D), or R_(D);

R₆ is H, OH, OR_(D), or R_(D);

R₇ is H, OH, OR_(D), or R_(D);

R₈ is OH, NH₂, or OR_(D);

R₉ is H, OH, or OR_(D);

X is —S—, —S(O)—, or —SO₂—; and

R_(D) is optionally substituted alkyl (e.g., C₁-C₆ alkyl), optionallysubstituted heteroalkyl (e.g., C₁-C₆ heteroalkyl), optionallysubstituted alkenyl (e.g., C₂-C₆ alkenyl), optionally substitutedheteroalkenyl (e.g., C₂-C₆ heteroalkenyl), optionally substitutedalkynyl (e.g., C₂-C₆ alkynyl), optionally substituted heteroalkynyl(e.g., C₂-C₆ heteroalkynyl), optionally substituted cycloalkyl,optionally substituted heterocycloalkyl, optionally substituted aryl, oroptionally substituted heteroaryl.

Amatoxins useful in conjunction with the compositions and methodsdescribed herein also include compounds according to formula (IIIB),below:

wherein R₁ is H, OH, or OR_(A);

R₂ is H, OH, or OR_(B);

R_(A) and R_(B), together with the oxygen atoms to which they are bound,combine to form an optionally substituted 5-membered heterocyclolalkylgroup;

R₃ is H or R_(D);

R₄ is H, OH, OR_(D), or R_(D);

R₅ is H, OH, OR_(D), or R_(D);

R₆ is H, OH, OR_(D), or R_(D);

R₇ is H, OH, OR_(D), or R_(D);

R₈ is OH, NH₂, or OR_(D);

R₉ is H, OH, or OR_(D);

X is —S—, —S(O)—, or —SO₂—; and

R_(D) is optionally substituted alkyl (e.g., C₁-C₆ alkyl), optionallysubstituted heteroalkyl (e.g., C₁-C₆ heteroalkyl), optionallysubstituted alkenyl (e.g., C₂-C₆ alkenyl), optionally substitutedheteroalkenyl (e.g., C₂-C₆ heteroalkenyl), optionally substitutedalkynyl (e.g., C₂-C₆ alkynyl), optionally substituted heteroalkynyl(e.g., C₂-C₆ heteroalkynyl), optionally substituted cycloalkyl,optionally substituted heterocycloalkyl, optionally substituted aryl, oroptionally substituted heteroaryl.

As described herein, amatoxins may be conjugated to an antibody,antigen-binding fragment thereof, or ligand, for instance, by way of alinker moiety. Exemplary methods of amatoxin conjugation and linkersuseful for such processes are described in the section entitled “Linkersfor chemical conjugation,” as well as in Table 2, below. Exemplarylinker-containing amatoxins useful for conjugation to an antibody,antigen-binding fragment, or ligand in accordance with the compositionsand methods described herein are shown in structural formulas (I), (IA),(IB) and (II), recited herein.

As used herein, the term “antibody” refers to an immunoglobulin moleculethat specifically binds to, or is immunologically reactive with, aparticular antigen, and includes polyclonal, monoclonal, geneticallyengineered, and otherwise modified forms of antibodies, including butnot limited to chimeric antibodies, humanized antibodies,heteroconjugate antibodies (e.g., bi- tri- and quad-specific antibodies,diabodies, triabodies, and tetrabodies), and antigen binding fragmentsof antibodies, including, for example, Fab′, F(ab′)₂, Fab, Fv, rIgG, andscFv fragments. Unless otherwise indicated, the term “monoclonalantibody” (mAb) is meant to include both intact molecules, as well asantibody fragments (including, for example, Fab and F(ab′)₂ fragments)that are capable of specifically binding to a target protein. As usedherein, the Fab and F(ab′)₂ fragments refer to antibody fragments thatlack the Fc fragment of an intact antibody. Examples of these antibodyfragments are described herein.

The term “antigen-binding fragment,” as used herein, refers to one ormore fragments of an antibody that retain the ability to specificallybind to a target antigen. The antigen-binding function of an antibodycan be performed by fragments of a full-length antibody. The antibodyfragments can be, for example, a Fab, F(ab′)₂, scFv, diabody, atriabody, an affibody, a nanobody, an aptamer, or a domain antibody.Examples of binding fragments encompassed of the term “antigen-bindingfragment” of an antibody include, but are not limited to: (i) a Fabfragment, a monovalent fragment consisting of the V_(L), V_(H), C_(L),and C_(H)1 domains; (ii) a F(ab′)₂ fragment, a bivalent fragmentcontaining two Fab fragments linked by a disulfide bridge at the hingeregion; (iii) a Fd fragment consisting of the V_(H) and C_(H)1 domains;(iv) a Fv fragment consisting of the V_(L) and V_(H) domains of a singlearm of an antibody, (v) a dAb including V_(H) and V_(L) domains; (vi) adAb fragment that consists of a V_(H) domain (see, e.g., Ward et al.,Nature 341:544-546, 1989); (vii) a dAb which consists of a V_(H) or aV_(L) domain; (viii) an isolated complementarity determining region(CDR); and (ix) a combination of two or more (e.g., two, three, four,five, or six) isolated CDRs which may optionally be joined by asynthetic linker. Furthermore, although the two domains of the Fvfragment, V_(L) and V_(H), are coded for by separate genes, they can bejoined, using recombinant methods, by a linker that enables them to bemade as a single protein chain in which the V_(L) and V_(H) regions pairto form monovalent molecules (known as single chain Fv (scFv); see, forexample, Bird et al., Science 242:423-426, 1988 and Huston et al., Proc.Natl. Acad. Sci. USA 85:5879-5883, 1988). These antibody fragments canbe obtained using conventional techniques known to those of skill in theart, and the fragments can be screened for utility in the same manner asintact antibodies. Antigen-binding fragments can be produced byrecombinant DNA techniques, enzymatic or chemical cleavage of intactimmunoglobulins, or, in certain cases, by chemical peptide synthesisprocedures known in the art.

As used herein, the term “anti-CD45 antibody” refers to a protein orpeptide-containing molecule that includes at least a portion of animmunoglobulin molecule, such as but not limited to at least onecomplementarity determining region (CDR) of a heavy or light chain or aligand binding portion thereof, a heavy chain or light chain variableregion, a heavy chain or light chain constant region, a frameworkregion, or any portion thereof, that is capable of specifically bindingto CD45 (for example, CD45RO). Anti-CD45 antibodies also includeantibody-like protein scaffolds, such as the tenth fibronectin type IIIdomain (¹⁰Fn3), which contains BC, DE, and FG structural loops similarin structure and solvent accessibility to antibody CDRs. The tertiarystructure of the ¹⁰Fn3 domain resembles that of the variable region ofthe IgG heavy chain, and one of skill in the art can graft, for example,the CDRs of an anti-CD45 monoclonal antibody onto the fibronectinscaffold by replacing residues of the BC, DE, and FG loops of ¹⁰Fn3 withresidues from the CDRH-1, CDRH-2, or CDRH-3 regions of an anti-CD45monoclonal antibody.

As used herein, the term “anti-CD135 antibody” refers to a protein orpeptide-containing molecule that includes at least a portion of animmunoglobulin molecule, such as but not limited to at least onecomplementarity determining region (CDR) of a heavy or light chain or aligand binding portion thereof, a heavy chain or light chain variableregion, a heavy chain or light chain constant region, a frameworkregion, or any portion thereof, that is capable of specifically bindingto CD135. Anti-CD135 antibodies also include antibody-like proteinscaffolds, such as the tenth fibronectin type III domain (¹⁰Fn3), whichcontains BC, DE, and FG structural loops similar in structure andsolvent accessibility to antibody CDRs. The tertiary structure of the¹⁰Fn3 domain resembles that of the variable region of the IgG heavychain, and one of skill in the art can graft, for example, the CDRs ofan anti-CD135 monoclonal antibody onto the fibronectin scaffold byreplacing residues of the BC, DE, and FG loops of ¹⁰Fn3 with residuesfrom the CDRH-1, CDRH-2, or CDRH-3 regions of an anti-CD135 monoclonalantibody.

As used herein, the term “anti-CD34 antibody” refers to a protein orpeptide-containing molecule that includes at least a portion of animmunoglobulin molecule, such as but not limited to at least onecomplementarity determining region (CDR) of a heavy or light chain or aligand binding portion thereof, a heavy chain or light chain variableregion, a heavy chain or light chain constant region, a frameworkregion, or any portion thereof, that is capable of specifically bindingto CD34. Anti-CD34 antibodies also include antibody-like proteinscaffolds, such as the tenth fibronectin type III domain (¹⁰Fn3), whichcontains BC, DE, and FG structural loops similar in structure andsolvent accessibility to antibody CDRs. The tertiary structure of the¹⁰Fn3 domain resembles that of the variable region of the IgG heavychain, and one of skill in the art can graft, for example, the CDRs ofan anti-CD34 monoclonal antibody onto the fibronectin scaffold byreplacing residues of the BC, DE, and FG loops of ¹⁰Fn3 with residuesfrom the CDRH-1, CDRH-2, or CDRH-3 regions of an anti-CD34 monoclonalantibody.

As used herein, the term “anti-CD90 antibody” refers to a protein orpeptide-containing molecule that includes at least a portion of animmunoglobulin molecule, such as but not limited to at least onecomplementarity determining region (CDR) of a heavy or light chain or aligand binding portion thereof, a heavy chain or light chain variableregion, a heavy chain or light chain constant region, a frameworkregion, or any portion thereof, that is capable of specifically bindingto CD90. Anti-CD90 antibodies also include antibody-like proteinscaffolds, such as the tenth fibronectin type III domain (¹⁰Fn3), whichcontains BC, DE, and FG structural loops similar in structure andsolvent accessibility to antibody CDRs. The tertiary structure of the¹⁰Fn3 domain resembles that of the variable region of the IgG heavychain, and one of skill in the art can graft, for example, the CDRs ofan anti-CD90 monoclonal antibody onto the fibronectin scaffold byreplacing residues of the BC, DE, and FG loops of ¹⁰Fn3 with residuesfrom the CDRH-1, CDRH-2, or CDRH-3 regions of an anti-CD90 monoclonalantibody.

As used herein, the term “anti-CD110 antibody” refers to a protein orpeptide-containing molecule that includes at least a portion of animmunoglobulin molecule, such as but not limited to at least onecomplementarity determining region (CDR) of a heavy or light chain or aligand binding portion thereof, a heavy chain or light chain variableregion, a heavy chain or light chain constant region, a frameworkregion, or any portion thereof, that is capable of specifically bindingto CD110. Anti-CD110 antibodies also include antibody-like proteinscaffolds, such as the tenth fibronectin type III domain (¹⁰Fn3), whichcontains BC, DE, and FG structural loops similar in structure andsolvent accessibility to antibody CDRs. The tertiary structure of the¹⁰Fn3 domain resembles that of the variable region of the IgG heavychain, and one of skill in the art can graft, for example, the CDRs ofan anti-CD110 monoclonal antibody onto the fibronectin scaffold byreplacing residues of the BC, DE, and FG loops of ¹⁰Fn3 with residuesfrom the CDRH-1, CDRH-2, or CDRH-3 regions of an anti-CD110 monoclonalantibody.

As used herein, the term “bispecific antibody” refers to, for example, amonoclonal, often a human or humanized antibody that is capable ofbinding at least two different antigens. For instance, one of thebinding specificities can be directed towards a hematopoietic stem cellsurface antigen, such as CD45 (e.g., CD45RO), CD135, CD34, CD90, orCD110, the other can be for a different hematopoietic stem cell surfaceantigen or another cell surface protein, such as a receptor or receptorsubunit involved in a signal transduction pathway that potentiates cellgrowth, among others.

As used herein, the term “complementarity determining region” (CDR)refers to a hypervariable region found both in the light chain and theheavy chain variable domains of an antibody. The more highly conservedportions of variable domains are referred to as framework regions (FRs).The amino acid positions that delineate a hypervariable region of anantibody can vary, depending on the context and the various definitionsknown in the art. Some positions within a variable domain may be viewedas hybrid hypervariable positions in that these positions can be deemedto be within a hypervariable region under one set of criteria whilebeing deemed to be outside a hypervariable region under a different setof criteria. One or more of these positions can also be found inextended hypervariable regions. The antibodies described herein maycontain modifications in these hybrid hypervariable positions. Thevariable domains of native heavy and light chains each contain fourframework regions that primarily adopt a β-sheet configuration,connected by three CDRs, which form loops that connect, and in somecases form part of, the β-sheet structure. The CDRs in each chain areheld together in close proximity by the framework regions in the orderFR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and, with the CDRs from the otherantibody chains, contribute to the formation of the target binding siteof antibodies (see Kabat et al., Sequences of Proteins of ImmunologicalInterest, National Institute of Health, Bethesda, Md., 1987). As usedherein, numbering of immunoglobulin amino acid residues is performedaccording to the immunoglobulin amino acid residue numbering system ofKabat et al., unless otherwise indicated.

As used herein, the terms “condition” and “conditioning” refer toprocesses by which a patient is prepared for receipt of a transplantcontaining hematopoietic stem cells. Such procedures promote theengraftment of a hematopoietic stem cell transplant (for instance, asinferred from a sustained increase in the quantity of viablehematopoietic stem cells within a blood sample isolated from a patientfollowing a conditioning procedure and subsequent hematopoietic stemcell transplantation. According to the methods described herein, apatient may be conditioned for hematopoietic stem cell transplanttherapy by administration to the patient of an antibody orantigen-binding fragment thereof capable of binding an antigen expressedby hematopoietic stem cells, such as CD45 (e.g., CD45RO), CD135, CD34,CD90, or CD110. As described herein, the antibody may be covalentlyconjugated to a cytotoxin so as to form a drug-antibody conjugate.Administration of an antibody, antigen-binding fragment thereof, ordrug-antibody conjugate capable of binding one or more of the foregoingantigens to a patient in need of hematopoietic stem cell transplanttherapy can promote the engraftment of a hematopoietic stem cell graft,for example, by selectively depleting endogenous hematopoietic stemcells, thereby creating a vacancy filled by an exogenous hematopoieticstem cell transplant.

As used herein, the term “conjugate” refers to a compound formed by thechemical bonding of a reactive functional group of one molecule, such asan antibody or antigen-binding fragment thereof, with an appropriatelyreactive functional group of another molecule, such as a cytotoxindescribed herein. Conjugates may include a linker between the twomolecules bound to one another. Examples of linkers that can be used forthe formation of a conjugate include peptide-containing linkers, such asthose that contain naturally occurring or non-naturally occurring aminoacids, such as D-amino acids. Linkers can be prepared using a variety ofstrategies described herein and known in the art. Depending on thereactive components therein, a linker may be cleaved, for example, byenzymatic hydrolysis, photolysis, hydrolysis under acidic conditions,hydrolysis under basic conditions, oxidation, disulfide reduction,nucleophilic cleavage, or organometallic cleavage (see, for example,Leriche et al., Bioorg. Med. Chem., 20:571-582, 2012).

As used herein, the term “coupling reaction” refers to a chemicalreaction in which two or more substituents suitable for reaction withone another react so as to form a chemical moiety that joins (e.g.,covalently) the molecular fragments bound to each substituent. Couplingreactions include those in which a reactive substituent bound to afragment that is a cytotoxin, such as a cytotoxin known in the art ordescribed herein, reacts with a suitably reactive substituent bound to afragment that is an antibody, antigen-binding fragment thereof, orligand, such as an antibody, antigen-binding fragment thereof, or ligandspecific for CD45 (such as CD45RO), CD135, CD34, CD90, and/or CD110known in the art or described herein. Examples of suitably reactivesubstituents include a nucleophile/electrophile pair (e.g., athiol/haloalkyl pair, an amine/carbonyl pair, or a thiol/α,β-unsaturatedcarbonyl pair, among others), a diene/dienophile pair (e.g., anazide/alkyne pair, among others), and the like. Coupling reactionsinclude, without limitation, thiol alkylation, hydroxyl alkylation,amine alkylation, amine condensation, amidation, esterification,disulfide formation, cycloaddition (e.g., [4+2] Diels-Aldercycloaddition, [3+2] Huisgen cycloaddition, among others), nucleophilicaromatic substitution, electrophilic aromatic substitution, and otherreactive modalities known in the art or described herein.

As used herein, “CRU (competitive repopulating unit)” refers to a unitof measure of long-term engrafting stem cells, which can be detectedafter in-vivo transplantation.

As used herein, the term “donor” refers to a human or animal from whichone or more cells are isolated prior to administration of the cells, orprogeny thereof, into a recipient. The one or more cells may be, forexample, a population of hematopoietic stem cells.

As used herein, the term “diabody” refers to a bivalent antibodycontaining two polypeptide chains, in which each polypeptide chainincludes V_(H) and V_(L) domains joined by a linker that is too short(e.g., a linker composed of five amino acids) to allow forintramolecular association of V_(H) and V_(L) domains on the samepeptide chain. This configuration forces each domain to pair with acomplementary domain on another polypeptide chain so as to form ahomodimeric structure. Accordingly, the term “triabody” refers totrivalent antibodies containing three peptide chains, each of whichcontains one V_(H) domain and one V_(L) domain joined by a linker thatis exceedingly short (e.g., a linker composed of 1-2 amino acids) topermit intramolecular association of V_(H) and V_(L) domains within thesame peptide chain. In order to fold into their native structures,peptides configured in this way typically trimerize so as to positionthe V_(H) and V_(L) domains of neighboring peptide chains spatiallyproximal to one another (see, for example, Holliger et al., Proc. Natl.Acad. Sci. USA 90:6444-48, 1993).

As used herein, a “dual variable domain immunoglobulin” (“DVD-Ig”)refers to an antibody that combines the target-binding variable domainsof two monoclonal antibodies via linkers to create a tetravalent,dual-targeting single agent (see, for example, Gu et al., Meth.Enzymol., 502:25-41, 2012).

As used herein, the term “endogenous” describes a substance, such as amolecule, cell, tissue, or organ (e.g., a hematopoietic stem cell or acell of hematopoietic lineage, such as a megakaryocyte, thrombocyte,platelet, erythrocyte, mast cell, myeoblast, basophil, neutrophil,eosinophil, microglial cell, granulocyte, monocyte, osteoclast,antigen-presenting cell, macrophage, dendritic cell, natural killercell, T-lymphocyte, or B-lymphocyte) that is found naturally in aparticular organism, such as a human patient.

As used herein, the term “engraftment potential” is used to refer to theability of hematopoietic stem and progenitor cells to repopulate atissue, whether such cells are naturally circulating or are provided bytransplantation. The term encompasses all events surrounding or leadingup to engraftment, such as tissue homing of cells and colonization ofcells within the tissue of interest. The engraftment efficiency or rateof engraftment can be evaluated or quantified using any clinicallyacceptable parameter as known to those of skill in the art and caninclude, for example, assessment of competitive repopulating units(CRU); incorporation or expression of a marker in tissue(s) into whichstem cells have homed, colonized, or become engrafted; or by evaluationof the progress of a subject through disease progression, survival ofhematopoietic stem and progenitor cells, or survival of a recipient.Engraftment can also be determined by measuring white blood cell countsin peripheral blood during a post-transplant period. Engraftment canalso be assessed by measuring recovery of marrow cells by donor cells ina bone marrow aspirate sample.

As used herein, the term “exogenous” describes a substance, such as amolecule, cell, tissue, or organ (e.g., a hematopoietic stem cell or acell of hematopoietic lineage, such as a megakaryocyte, thrombocyte,platelet, erythrocyte, mast cell, myeoblast, basophil, neutrophil,eosinophil, microglial cell, granulocyte, monocyte, osteoclast,antigen-presenting cell, macrophage, dendritic cell, natural killercell, T-lymphocyte, or B-lymphocyte) that is not found naturally in aparticular organism, such as a human patient. Exogenous substancesinclude those that are provided from an external source to an organismor to cultured matter extracted therefrom.

As used herein, the term “framework region” or “FW region” includesamino acid residues that are adjacent to the CDRs of an antibody orantigen-binding fragment thereof. FW region residues may be present in,for example, human antibodies, humanized antibodies, monoclonalantibodies, antibody fragments, Fab fragments, single chain antibodyfragments, scFv fragments, antibody domains, and bispecific antibodies,among others.

As used herein, the term “hematopoietic stem cells” (“HSCs”) refers toimmature blood cells having the capacity to self-renew and todifferentiate into mature blood cells containing diverse lineagesincluding but not limited to granulocytes (e.g., promyelocytes,neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes,erythrocytes), thrombocytes (e.g., megakaryoblasts, platelet producingmegakaryocytes, platelets), monocytes (e.g., monocytes, macrophages),dendritic cells, microglia, osteoclasts, and lymphocytes (e.g., NKcells, B-cells and T-cells). Such cells may include CD34+ cells. CD34+cells are immature cells that express the CD34 cell surface marker. Inhumans, CD34+ cells are believed to include a subpopulation of cellswith the stem cell properties defined above, whereas in mice, HSCs areCD34-. In addition, HSCs also refer to long term repopulating HSCs(LT-HSC) and short term repopulating HSCs (ST-HSC). LT-HSCs and ST-HSCsare differentiated, based on functional potential and on cell surfacemarker expression. For example, human HSCs are CD34+, CD38−, CD45RA−,CD90+, CD49F+, and lin− (negative for mature lineage markers includingCD2, CD3, CD4, CD7, CD8, CD10, CD11B, CD19, CD20, CD56, CD235A). Inmice, bone marrow LT-HSCs are CD34−, SCA-1+, C-kit+, CD135−,Slamfl/CD150+, CD48−, and lin− (negative for mature lineage markersincluding Ter119, CD11b, Gr1, CD3, CD4, CD8, B220, IL7ra), whereasST-HSCs are CD34+, SCA-1+, C-kit+, CD135−, Slamfl/CD150+, and lin−(negative for mature lineage markers including Ter119, CD11b, Gr1, CD3,CD4, CD8, B220, IL7ra). In addition, ST-HSCs are less quiescent and moreproliferative than LT-HSCs under homeostatic conditions. However, LT-HSChave greater self renewal potential (i.e., they survive throughoutadulthood, and can be serially transplanted through successiverecipients), whereas ST-HSCs have limited self renewal (i.e., theysurvive for only a limited period of time, and do not possess serialtransplantation potential). Any of these HSCs can be used in the methodsdescribed herein. ST-HSCs are particularly useful because they arehighly proliferative and thus, can more quickly give rise todifferentiated progeny.

As used herein, the term “hematopoietic stem cell functional potential”refers to the functional properties of hematopoietic stem cells whichinclude 1) multi-potency (which refers to the ability to differentiateinto multiple different blood lineages including, but not limited to,granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils),erythrocytes (e.g., reticulocytes, erythrocytes), thrombocytes (e.g.,megakaryoblasts, platelet producing megakaryocytes, platelets),monocytes (e.g., monocytes, macrophages), dendritic cells, microglia,osteoclasts, and lymphocytes (e.g., NK cells, B-cells and T-cells), 2)self-renewal (which refers to the ability of hematopoietic stem cells togive rise to daughter cells that have equivalent potential as the mothercell, and further that this ability can repeatedly occur throughout thelifetime of an individual without exhaustion), and 3) the ability ofhematopoietic stem cells or progeny thereof to be reintroduced into atransplant recipient whereupon they home to the hematopoietic stem cellniche and re-establish productive and sustained hematopoiesis.

As used herein, the term “human antibody” refers to an antibody in whichsubstantially every part of the protein (for example, all CDRs,framework regions, C_(L), C_(H) domains (e.g., C_(H)1, C_(H)2, C_(H)3),hinge, and V_(L) and V_(H) domains) is substantially non-immunogenic inhumans, with only minor sequence changes or variations. A human antibodycan be produced in a human cell (for example, by recombinant expression)or by a non-human animal or a prokaryotic or eukaryotic cell that iscapable of expressing functionally rearranged human immunoglobulin (suchas heavy chain and/or light chain) genes. When a human antibody is asingle chain antibody, it can include a linker peptide that is not foundin native human antibodies. For example, an Fv can contain a linkerpeptide, such as two to about eight glycine or other amino acidresidues, which connects the variable region of the heavy chain and thevariable region of the light chain. Such linker peptides are consideredto be of human origin. Human antibodies can be made by a variety ofmethods known in the art including phage display methods using antibodylibraries derived from human immunoglobulin sequences. Human antibodiescan also be produced using transgenic mice that are incapable ofexpressing functional endogenous immunoglobulins, but which can expresshuman immunoglobulin genes (see, for example, PCT Publication Nos. WO1998/24893; WO 1992/01047; WO 1996/34096; WO 1996/33735; U.S. Pat. Nos.5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806;5,814,318; 5,885,793; 5,916,771; and 5,939,598).

As used herein, the term “humanized” antibody refers to a non-humanantibody that contains minimal sequences derived from non-humanimmunoglobulin. In general, a humanized antibody contains substantiallyall of at least one, and typically two, variable domains, in which allor substantially all of the CDR regions correspond to those of anon-human immunoglobulin. All or substantially all of the FW regions mayalso be those of a human immunoglobulin sequence. The humanized antibodycan also contain at least a portion of an immunoglobulin constant region(Fc), typically that of a human immunoglobulin consensus sequence.Methods of antibody humanization are known in the art and have beendescribed, for example, in Riechmann et al., Nature 332:323-7, 1988;U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,761; 5,693,762; and6,180,370.

As used herein, patients that are “in need of” a hematopoietic stem celltransplant include patients that exhibit a defect or deficiency in oneor more blood cell types, as well as patients having a stem celldisorder, autoimmune disease, cancer, or other pathology describedherein. Hematopoietic stem cells generally exhibit 1) multi-potency, andcan thus differentiate into multiple different blood lineages including,but not limited to, granulocytes (e.g., promyelocytes, neutrophils,eosinophils, basophils), erythrocytes (e.g., reticulocytes,erythrocytes), thrombocytes (e.g., megakaryoblasts, platelet producingmegakaryocytes, platelets), monocytes (e.g., monocytes, macrophages),dendritic cells, microglia, osteoclasts, and lymphocytes (e.g., NKcells, B-cells and T-cells), 2) self-renewal, and can thus give rise todaughter cells that have equivalent potential as the mother cell, and 3)the ability to be reintroduced into a transplant recipient whereuponthey home to the hematopoietic stem cell niche and re-establishproductive and sustained hematopoiesis. Hematopoietic stem cells canthus be administered to a patient defective or deficient in one or morecell types of the hematopoietic lineage in order to re-constitute thedefective or deficient population of cells in vivo. For example, thepatient may be suffering from cancer, and the deficiency may be causedby administration of a chemotherapeutic agent or other medicament thatdepletes, either selectively or non-specifically, the cancerous cellpopulation. Additionally or alternatively, the patient may be sufferingfrom a hemoglobinopathy (e.g., a non-malignant hemoglobinopathy), suchas sickle cell anemia, thalassemia, Fanconi anemia, aplastic anemia, andWiskott-Aldrich syndrome. The subject may be one that is suffering fromadenosine deaminase severe combined immunodeficiency (ADA SCID),HIV/AIDS, metachromatic leukodystrophy, Diamond-Blackfan anemia, andSchwachman-Diamond syndrome. The subject may have or be affected by aninherited blood disorder (e.g., sickle cell anemia) or an autoimmunedisorder. Additionally or alternatively, the subject may have or beaffected by a malignancy, such as neuroblastoma or a hematologic cancer.For instance, the subject may have a leukemia, lymphoma, or myeloma. Insome embodiments, the subject has acute myeloid leukemia, acute lymphoidleukemia, chronic myeloid leukemia, chronic lymphoid leukemia, multiplemyeloma, diffuse large B-cell lymphoma, or non-Hodgkin's lymphoma. Insome embodiments, the subject has myelodysplastic syndrome. In someembodiments, the subject has an autoimmune disease, such as scleroderma,multiple sclerosis, ulcerative colitis, Crohn's disease, Type 1diabetes, or another autoimmune pathology described herein. In someembodiments, the subject is in need of chimeric antigen receptor T-cell(CART) therapy. In some embodiments, the subject has or is otherwiseaffected by a metabolic storage disorder. The subject may suffer orotherwise be affected by a metabolic disorder selected from the groupconsisting of glycogen storage diseases, mucopolysaccharidoses,Gaucher's Disease, Hurlers Disease, sphingolipidoses, metachromaticleukodystrophy, or any other diseases or disorders which may benefitfrom the treatments and therapies disclosed herein and including,without limitation, severe combined immunodeficiency, Wiscott-Aldrichsyndrome, hyper immunoglobulin M (IgM) syndrome, Chediak-Higashidisease, hereditary lymphohistiocytosis, osteopetrosis, osteogenesisimperfecta, storage diseases, thalassemia major, sickle cell disease,systemic sclerosis, systemic lupus erythematosus, multiple sclerosis,juvenile rheumatoid arthritis and those diseases, or disorders describedin “Bone Marrow Transplantation for Non-Malignant Disease,” ASHEducation Book, 1:319-338 (2000), the disclosure of which isincorporated herein by reference in its entirety as it pertains topathologies that may be treated by administration of hematopoietic stemcell transplant therapy. Additionally or alternatively, a patient “inneed of” a hematopoietic stem cell transplant may one that is or is notsuffering from one of the foregoing pathologies, but nonethelessexhibits a reduced level (e.g., as compared to that of an otherwisehealthy subject) of one or more endogenous cell types within thehematopoietic lineage, such as megakaryocytes, thrombocytes, platelets,erythrocytes, mast cells, myeoblasts, basophils, neutrophils,eosinophils, microglia, granulocytes, monocytes, osteoclasts,antigen-presenting cells, macrophages, dendritic cells, natural killercells, T-lymphocytes, and B-lymphocytes. One of skill in the art canreadily determine whether one's level of one or more of the foregoingcell types, or other blood cell type, is reduced with respect to anotherwise healthy subject, for instance, by way of flow cytometry andfluorescence activated cell sorting (FACS) methods, among otherprocedures, known in the art.

As used herein, the term “monoclonal antibody” refers to an antibodythat is derived from a single clone, including any eukaryotic,prokaryotic, or phage clone, and not the method by which it is produced.

As used herein, the term “recipient” refers to a patient that receives atransplant, such as a transplant containing a population ofhematopoietic stem cells. The transplanted cells administered to arecipient may be, e.g., autologous, syngeneic, or allogeneic cells.

As used herein, the term “sample” refers to a specimen (e.g., blood,blood component (e.g., serum or plasma), urine, saliva, amniotic fluid,cerebrospinal fluid, tissue (e.g., placental or dermal), pancreaticfluid, chorionic villus sample, and cells) taken from a subject.

As used herein, the term “scFv” refers to a single chain Fv antibody inwhich the variable domains of the heavy chain and the light chain froman antibody have been joined to form one chain. scFv fragments contain asingle polypeptide chain that includes the variable region of anantibody light chain (V_(L)) (e.g., CDR-L1, CDR-L2, and/or CDR-L3) andthe variable region of an antibody heavy chain (V_(H)) (e.g., CDR-H1,CDR-H2, and/or CDR-H3) separated by a linker. The linker that joins theV_(L) and V_(H) regions of a scFv fragment can be a peptide linkercomposed of proteinogenic amino acids. Alternative linkers can be usedto so as to increase the resistance of the scFv fragment to proteolyticdegradation (for example, linkers containing D-amino acids), in order toenhance the solubility of the scFv fragment (for example, hydrophiliclinkers such as polyethylene glycol-containing linkers or polypeptidescontaining repeating glycine and serine residues), to improve thebiophysical stability of the molecule (for example, a linker containingcysteine residues that form intramolecular or intermolecular disulfidebonds), or to attenuate the immunogenicity of the scFv fragment (forexample, linkers containing glycosylation sites). It will also beunderstood by one of ordinary skill in the art that the variable regionsof the scFv molecules described herein can be modified such that theyvary in amino acid sequence from the antibody molecule from which theywere derived. For example, nucleotide or amino acid substitutionsleading to conservative substitutions or changes at amino acid residuescan be made (e.g., in CDR and/or framework residues) so as to preserveor enhance the ability of the scFv to bind to the antigen recognized bythe corresponding antibody.

As used herein, the terms “subject” and “patient” refer to an organism,such as a human, that receives treatment for a particular disease orcondition as described herein. For instance, a patient, such as a humanpatient, may receive treatment prior to hematopoietic stem celltransplant therapy in order to promote the engraftment of exogenoushematopoietic stem cells.

As used herein, the phrase “substantially cleared from the blood” refersto a point in time following administration of a therapeutic agent (suchas an anti-CD45, anti-CD135, anti-CD34, anti-CD90, or anti-CD110antibody, antigen-binding fragment thereof, or ligand) to a patient whenthe concentration of the therapeutic agent in a blood sample isolatedfrom the patient is such that the therapeutic agent is not detectable byconventional means (for instance, such that the therapeutic agent is notdetectable above the noise threshold of the device or assay used todetect the therapeutic agent). A variety of techniques known in the artcan be used to detect antibodies, antibody fragments, and proteinligands, such as ELISA-based detection assays known in the art ordescribed herein. Additional assays that can be used to detectantibodies, antibody fragments, and protein ligands includeimmunoprecipitation techniques and immunoblot assays, among others knownin the art.

As used herein, the phrase “stem cell disorder” broadly refers to anydisease, disorder, or condition that may be treated or cured byconditioning a subjects target tissues, and/or by ablating an endogenousstem cell population in a target tissue (e.g., ablating an endogenoushematopoietic stem or progenitor cell population from a subject's bonemarrow tissue) and/or by engrafting or transplanting stem cells in asubjects target tissues. For example, Type I diabetes has been shown tobe cured by hematopoietic stem cell transplant and may benefit fromconditioning in accordance with the compositions and methods describedherein. Additional disorders that can be treated using the compositionsand methods described herein include, without limitation, sickle cellanemia, thalassemias, Fanconi anemia, aplastic anemia, Wiskott-Aldrichsyndrome, ADA SCID, HIV/AIDS, metachromatic leukodystrophy,Diamond-Blackfan anemia, and Schwachman-Diamond syndrome. Additionaldiseases that may be treated using the patient conditioning and/orhematopoietic stem cell transplant methods described herein infludeinherited blood disorders (e.g., sickle cell anemia) and autoimmunedisorders, such as scleroderma, multiple sclerosis, ulcerative colitis,and Chrohn's disease. Additional diseases that may be treated using theconditioning and/or transplantation methods described herein include amalignancy, such as a neuroblastoma or a hematologic cancers, such asleukemia, lymphoma, and myeloma. For instance, the cancer may be acutemyeloid leukemia, acute lymphoid leukemia, chronic myeloid leukemia,chronic lymphoid leukemia, multiple myeloma, diffuse large B-celllymphoma, or non-Hodgkin's lymphoma. Additional diseases treatable usingthe conditioning and/or transplantation methods described herein includemyelodysplastic syndrome. In some embodiments, the subject has or isotherwise affected by a metabolic storage disorder. For example, thesubject may suffer or otherwise be affected by a metabolic disorderselected from the group consisting of glycogen storage diseases,mucopolysaccharidoses, Gaucher's Disease, Hurlers Disease,sphingolipidoses, metachromatic leukodystrophy, or any other diseases ordisorders which may benefit from the treatments and therapies disclosedherein and including, without limitation, severe combinedimmunodeficiency, Wiscott-Aldrich syndrome, hyper immunoglobulin M (IgM)syndrome, Chediak-Higashi disease, hereditary lymphohistiocytosis,osteopetrosis, osteogenesis imperfecta, storage diseases, thalassemiamajor, sickle cell disease, systemic sclerosis, systemic lupuserythematosus, multiple sclerosis, juvenile rheumatoid arthritis andthose diseases, or disorders described in “Bone Marrow Transplantationfor Non-Malignant Disease,” ASH Education Book, 1:319-338 (2000), thedisclosure of which is incorporated herein by reference in its entiretyas it pertains to pathologies that may be treated by administration ofhematopoietic stem cell transplant therapy.

As used herein, the terms “treat” or “treatment” refer to therapeutictreatment, in which the object is to prevent or slow down (lessen) anundesired physiological change or disorder or to promote a beneficialphenotype in the patient being treated. Beneficial or desired clinicalresults include, but are not limited to, promoting the engraftment ofexogenous hematopoietic cells in a patient following antibodyconditioning therapy as described herein and subsequent hematopoieticstem cell transplant therapy. Additional beneficial results include anincrease in the cell count or relative concentration of hematopoieticstem cells in a patient in need of a hematopoietic stem cell transplantfollowing conditioning therapy and subsequent administration of anexogenous hematopoietic stem cell graft to the patient. Beneficialresults of therapy described herein may also include an increase in thecell count or relative concentration of one or more cells ofhematopoietic lineage, such as a megakaryocyte, thrombocyte, platelet,erythrocyte, mast cell, myeoblast, basophil, neutrophil, eosinophil,microglial cell, granulocyte, monocyte, osteoclast, antigen-presentingcell, macrophage, dendritic cell, natural killer cell, T-lymphocyte, orB-lymphocyte, following conditioning therapy and subsequenthematopoietic stem cell transplant therapy. Additional beneficialresults may include the reduction in quantity of a disease-causing cellpopulation, such as a population of cancer cells (e.g., CD45+, CD135+,CD34+, CD90+, or CD110+ leukemic cells) or autoimmune cells (e.g.,CD45+, CD135+, CD34+, CD90+, or CD110+ autoimmune lymphocytes, such as aCD45+, CD135+, CD34+, CD90+, or CD110+ T-cell that expresses a T-cellreceptor that cross-reacts with a self antigen).

As used herein, the terms “variant” and “derivative” are usedinterchangeably and refer to naturally-occurring, synthetic, andsemi-synthetic analogues of a compound, peptide, protein, or othersubstance described herein. A variant or derivative of a compound,peptide, protein, or other substance described herein may retain orimprove upon the biological activity of the original material.

As used herein, the term “vector” includes a nucleic acid vector, suchas a plasmid, a DNA vector, a plasmid, a RNA vector, virus, or othersuitable replicon. Expression vectors described herein may contain apolynucleotide sequence as well as, for example, additional sequenceelements used for the expression of proteins and/or the integration ofthese polynucleotide sequences into the genome of a mammalian cell.Certain vectors that can be used for the expression of antibodies andantibody fragments of the invention include plasmids that containregulatory sequences, such as promoter and enhancer regions, whichdirect gene transcription. Other useful vectors for expression ofantibodies and antibody fragments contain polynucleotide sequences thatenhance the rate of translation of these genes or improve the stabilityor nuclear export of the mRNA that results from gene transcription.These sequence elements may include, for example, 5′ and 3′ untranslatedregions and a polyadenylation signal site in order to direct efficienttranscription of the gene carried on the expression vector. Theexpression vectors described herein may also contain a polynucleotideencoding a marker for selection of cells that contain such a vector.Examples of a suitable marker include genes that encode resistance toantibiotics, such as ampicillin, chloramphenicol, kanamycin, andnourseothricin.

As used herein, the term “alkyl” refers to a straight- or branched-chainalkyl group having, for example, from 1 to 20 carbon atoms in the chain.Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl,butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, tert-pentyl,hexyl, isohexyl, and the like.

As used herein, the term “alkylene” refers to a straight- orbranched-chain divalent alkyl group. The divalent positions may be onthe same or different atoms within the alkyl chain. Examples of alkyleneinclude methylene, ethylene, propylene, isopropylene, and the like.

As used herein, the term “heteroalkyl” refers to a straight orbranched-chain alkyl group having, for example, from 1 to 20 carbonatoms in the chain, and further containing one or more heteroatoms(e.g., oxygen, nitrogen, or sulfur, among others) in the chain.

As used herein, the term “heteroalkylene” refers to a straight- orbranched-chain divalent heteroalkyl group. The divalent positions may beon the same or different atoms within the heteroalkyl chain. Thedivalent positions may be one or more heteroatoms.

As used herein, the term “alkenyl” refers to a straight- orbranched-chain alkenyl group having, for example, from 2 to 20 carbonatoms in the chain. Examples of alkenyl groups include vinyl, propenyl,isopropenyl, butenyl, tert-butylenyl, hexenyl, and the like.

As used herein, the term “alkenylene” refers to a straight- orbranched-chain divalent alkenyl group. The divalent positions may be onthe same or different atoms within the alkenyl chain. Examples ofalkenylene include ethenylene, propenylene, isopropenylene, butenylene,and the like.

As used herein, the term “heteroalkenyl” refers to a straight- orbranched-chain alkenyl group having, for example, from 2 to 20 carbonatoms in the chain, and further containing one or more heteroatoms(e.g., oxygen, nitrogen, or sulfur, among others) in the chain.

As used herein, the term “heteroalkenylene” refers to a straight- orbranched-chain divalent heteroalkenyl group. The divalent positions maybe on the same or different atoms within the heteroalkenyl chain. Thedivalent positions may be one or more heteroatoms.

As used herein, the term “alkynyl” refers to a straight- orbranched-chain alkynyl group having, for example, from 2 to 20 carbonatoms in the chain. Examples of alkynyl groups include propargyl,butynyl, pentynyl, hexynyl, and the like.

As used herein, the term “alkynylene” refers to a straight- orbranched-chain divalent alkynyl group. The divalent positions may be onthe same or different atoms within the alkynyl chain.

As used herein, the term “heteroalkynyl” refers to a straight- orbranched-chain alkynyl group having, for example, from 2 to 20 carbonatoms in the chain, and further containing one or more heteroatoms(e.g., oxygen, nitrogen, or sulfur, among others) in the chain.

As used herein, the term “heteroalkynylene” refers to a straight- orbranched-chain divalent heteroalkynyl group. The divalent positions maybe on the same or different atoms within the heteroalkynyl chain. Thedivalent positions may be one or more heteroatoms.

As used herein, the term “cycloalkyl” refers to a monocyclic, or fused,bridged, or spiro polycyclic ring structure that is saturated and has,for example, from 3 to 12 carbon ring atoms. Examples of cycloalkylgroups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl, cyclooctyl, bicyclo[3.1.0]hexane, and the like.

As used herein, the term “cycloalkylene” refers to a divalent cycloalkylgroup. The divalent positions may be on the same or different atomswithin the ring structure. Examples of cycloalkylene includecyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, and thelike.

As used herein, the term “heterocyloalkyl” refers to a monocyclic, orfused, bridged, or spiro polycyclic ring structure that is saturated andhas, for example, from 3 to 12 ring atoms per ring structure selectedfrom carbon atoms and heteroatoms selected from, e.g., nitrogen, oxygen,and sulfur, among others. The ring structure may contain, for example,one or more oxo groups on carbon, nitrogen, or sulfur ring members.

As used herein, the term “heterocycloalkylene” refers to a divalentheterocyclolalkyl group. The divalent positions may be on the same ordifferent atoms within the ring structure. The divalent positions may beone or more heteroatoms.

As used herein, the term “aryl” refers to a monocyclic or multicyclicaromatic ring system containing, for example, from 6 to 19 carbon atoms.Aryl groups include, but are not limited to, phenyl, fluorenyl,naphthyl, and the like.

As used herein, the term “arylene” refers to a divalent aryl group. Thedivalent positions may be on the same or different atoms.

As used herein, the term “heteroaryl” refers to a monocyclicheteroaromatic, or a bicyclic or a tricyclic fused-ring heteroaromaticgroup. Heteroaryl groups include pyridyl, pyrrolyl, furyl, thienyl,imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyrazolyl,1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadia-zolyl,1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, 1,3,4-triazinyl, 1,2,3-triazinyl,benzofuryl, [2,3-dihydro]benzofuryl, isobenzofuryl, benzothienyl,benzotriazolyl, isobenzothienyl, indolyl, isoindolyl, 3H-indolyl,benzimidazolyl, imidazo[1,2-a]pyridyl, benzothiazolyl, benzoxazolyl,quinolizinyl, quinazolinyl, pthalazinyl, quinoxalinyl, cinnolinyl,napthyridinyl, pyrido[3,4-b]pyridyl, pyrido[3,2-b]pyridyl,pyrido[4,3-b]pyridyl, quinolyl, isoquinolyl, tetrazolyl,5,6,7,8-tetrahydroquinolyl, 5,6,7,8-tetrahydroisoquinolyl, purinyl,pteridinyl, carbazolyl, xanthenyl, benzoquinolyl, and the like.

As used herein, the term “heteroarylene” refers to a divalent heteroarylgroup. The divalent positions may be on the same or different atoms. Thedivalent positions may be one or more heteroatoms.

Unless otherwise constrained by the definition of the individualsubstituent, the foregoing chemical moieties, such as “alkyl”,“alkylene”, “heteroalkyl”, “heteroalkylene”, “alkenyl”, “alkenylene”,“heteroalkenyl”, “heteroalkenylene”, “alkynyl”, “alkynylene”,“heteroalkynyl”, “heteroalkynylene”, “cycloalkyl”, “cycloalkylene”,“heterocyclolalkyl”, heterocycloalkylene”, “aryl,” “arylene”,“heteroaryl”, and “heteroarylene” groups can optionally be substitutedwith, for example, from 1 to 5 substituents selected from the groupconsisting of alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl,alkyl aryl, alkyl heteroaryl, alkyl cycloalkyl, alkyl heterocycloalkyl,amino, ammonium, acyl, acyloxy, acylamino, aminocarbonyl,alkoxycarbonyl, ureido, carbamate, aryl, heteroaryl, sulfinyl, sulfonyl,alkoxy, sulfanyl, halogen, carboxy, trihalomethyl, cyano, hydroxy,mercapto, nitro, and the like. The substitution may include situationsin which neighboring substituents have undergone ring closure, such asring closure of vicinal functional substituents, to form, for instance,lactams, lactones, cyclic anhydrides, acetals, hemiacetals, thioacetals,aminals, and hemiaminals, formed by ring closure, for example, tofurnish a protecting group.

Antibodies that Recognize Hematopoietic Cell Antigens

The present invention is based in part on the discovery that antibodies,antigen-binding fragments thereof, and ligands capable of binding CD45,CD135, CD34, CD90, or CD110 can be used as therapeutic agents to (i)directly treat cancers and autoimmune diseases characterized by cellsthat express one or more of these antigens and (ii) promote theengraftment of transplanted hematopoietic stem cells in a patient inneed of transplant therapy. These therapeutic activities can be caused,for instance, by the binding of antibodies, antigen-binding fragmentsthereof, and/or ligands to one or more of the foregoing antigensexpressed on the surface of a cell, such as a cancer cell, autoimmunecell, or hematopoietic stem cell and subsequently inducing cell death.The depletion of endogenous hematopoietic stem cells can provide a nichetoward which transplanted hematopoietic stem cells can home, andsubsequently establish productive hematopoiesis. In this way,transplanted hematopoietic stem cells may successfully engraft in apatient, such as human patient suffering from a stem cell disorderdescribed herein. The following sections describe each of these antigensas well as antibodies and antigen-binding fragments thereof capable ofbinding these targets.

Anti-CD45 Antibodies

Antibodies and antigen-binding fragments capable of binding human CD45(mRNA NCBI Reference Sequence: NM_080921.3, Protein NCBI ReferenceSequence: NP_563578.2), including those capable of binding the isoformCD45RO, can be used in conjunction with the compositions and methodsdisclosed herein, such as to promote engraftment of hematopoietic stemcell grafts in a patient in need of hematopoietic stem cell transplanttherapy. Multiple isoforms of CD45 arise from the alternative splicingof 34 exons in the primary transcript. Splicing of exons 4, 5, 6, andpotentially 7 give rise to multiple CD45 variations. Selective exonexpression is observed in the CD45 isoforms described in Table 1, below.

TABLE 1 Exon expression in various CD45 isoforms CD45 Isoform ExonExpression Pattern CD45RA Expresses exon 4 only CD45RB Expresses exon 5only CD45RC Expresses exon 6 only CD45RO Does not express exons 4-6

Alternative splicing can result in individual exons or combinations ofexons expressed in various isoforms of the CD45 protein (for example,CD45RA, CD45RAB, CD45RABC). In contrast, CD45RO lacks expression ofexons 4-6 and is generated from a combination of exons 1-3 and 7-34.There is evidence that exon 7 can also be excluded from the protein,resulting in splicing together of exons 1-3 and 8-34. This protein,designated E3-8, has been detected at the mRNA level but has not beencurrently identified by flow cytometry.

CD45RO is currently the only known CD45 isoform expressed onhematopoietic stem cells. CD45RA and CD45RABC have not been detected orare excluded from the phenotype of hematopoietic stem cells. There isevidence from studies conducted in mice that CD45RB is expressed onfetal hematopoietic stem cells, but it is not present on adult bonemarrow hematopoietic stem cells. Notably, CD45RC has a high rate ofpolymorphism in exon 6 found within Asian populations (a polymorphism atexon 6 in CD45RC is found in approximately 25% of the Japanesepopulation). This polymorphism leads to high expression of CD45RO anddecreased levels of CD45RA, CD45RB, and CD45RC. Additionally, CD45RAvariants (such as CD45RAB and CD45RAC) exhibit a polymorphism in exon 4that has been associated with autoimmune disease.

The presence of CD45RO on hematopoietic stem cells and its comparativelylimited expression on other immune cells (such as T and B lymphocytesubsets and various myeloid cells) renders CD45RO a particularlywell-suited target for conditioning therapy for patients in need of ahematopoietic stem cell transplant. As CD45RO only lacks expression ofexons 4, 5, and 6, its use as an immunogen enables the screening of panCD45 Abs and CD45RO-specific antibodies.

Anti-CD45 antibodies that can be used in conjunction with the patientconditioning methods described herein include, for example, theanti-CD45 antibody clone HI30, which is commercially available fromBIOLEGEND® (San Diego, Calif.), as well as humanized variants thereof.Humanization of antibodies can be performed by replacing frameworkresidues and constant region residues of a non-human antibody with thoseof a germline human antibody according to procedures known in the art(as described, for instance, in Example 7, below). Additional anti-CD45antibodies that can be used in conjunction with the methods describedherein include the anti-CD45 antibodies ab10558, EP322Y, MEM-28,ab10559, 0.N.125, F10-89-4, Hle-1, 2611, YTH24.5, PD7/26/16, F10-89-4,1B7, ab154885, B-A11, phosphor S1007, ab170444, EP350, Y321, GA90, D3/9,X1 6/99, and LT45, which are commercially available from ABCAM®(Cambridge, Mass.), as well as humanized variants thereof. Furtheranti-CD45 antibodies that may be used in conjunction with the patientconditioning procedures described herein include anti-CD45 antibodyHPA000440, which is commercially available from SIGMA-ALDRICH® (St.Louis, Mo.), and humanized variants thereof. Additional anti-CD45antibodies that can be used in conjunction with the patient conditioningmethods described herein include murine monoclonal antibody BC8, whichis described, for instance, in Matthews et al., Blood 78:1864-1874,1991, the disclosure of which is incorporated herein by reference as itpertains to anti-CD45 antibodies, as well as humanized variants thereof.Further anti-CD45 antibodies that can be used in conjunction with themethods described herein include monoclonal antibody YAML568, which isdescribed, for instance, in Glatting et al., J. Nucl. Med. 8:1335-1341,2006, the disclosure of which is incorporated herein by reference as itpertains to anti-CD45 antibodies, as well as humanized variants thereof.Additional anti-CD45 antibodies that can be used in conjunction with thepatient conditioning procedures described herein include monoclonalantibodies YTH54.12 and YTH25.4, which are described, for instance, inBrenner et al., Ann. N.Y. Acad. Sci. 996:80-88, 2003, the disclosure ofwhich is incorporated herein by reference as it pertains to anti-CD45antibodies, as well as humanized variants thereof. Additional anti-CD45antibodies for use with the patient conditioning methods describedherein include UCHL1, 2H4, SN130, MD4.3, MBI, and MT2, which aredescribed, for instance, in Brown et al., Immunology 64:331-336, 1998,the disclosure of which is incorporated herein by reference as itpertains to anti-CD45 antibodies, as well as humanized variants thereof.Additional anti-CD45 antibodies that can be used in conjunction with themethods described herein include those produced and released fromAmerican Type Culture Collection (ATCC) Accession Nos. RA3-6132,RA3-2C2, and TIB122, as well as monoclonal antibodies C363.16A, and13/2, which are described, for instance, in Johnson et al., J. Exp. Med.169:1179-1184, 1989, the disclosure of which is incorporated herein byreference as it pertains to anti-CD45 antibodies, as well as humanizedvariants thereof. Further anti-CD45 antibodies that can be used inconjunction with the patient conditioning methods described hereininclude the monoclonal antibodies AHN-12.1, AHN-12, AHN-12.2, AHN-12.3,AHN-12.4, HLe-1, and KC56(T200), which are described, for instance, inHarvath et al., J. Immunol. 146:949-957, 1991, the disclosure of whichis incorporated herein by reference as it pertains to anti-CD45antibodies, as well as humanized variants thereof.

Additional anti-CD45 antibodies that can be used in conjunction with thepatient conditioning methods described herein include those described,for example, in U.S. Pat. No. 7,265,212 (which describes, e.g.,anti-CD45 antibodies 39E11, 16C9, and 1G10, among other clones); U.S.Pat. No. 7,160,987 (which describe, e.g., anti-CD45 antibodies producedand released by ATCC Accession No. HB-11873, such as monoclonal antibody6G3); and U.S. Pat. No. 6,099,838 (which describes, e.g., anti-CD45antibody MT3, as well as antibodies produced and released by ATCCAccession Nos. HB220 (also designated MB23G2) and HB223), as well as US2004/0096901 and US 2008/0003224 (which describes, e.g., anti-CD45antibodies produced and released by ATCC Accession No. PTA-7339, such asmonoclonal antibody 17.1), the disclosures of each of which areincorporated herein by reference as they pertain to anti-CD45antibodies.

Further anti-CD45 antibodies that can be used in conjunction with thepatient conditioning methods described herein include antibodiesproduced and released from ATCC Accession Nos. MB4B4, MB23G2, 14.8, GAP8.3, 74-9-3, I/24.D6, 9.4, 4B2, M1/9.3.4.HL.2, as well as humanizedand/or affinity-matured variants thereof. Affinity maturation can beperformed, for instance, using in vitro display techniques describedherein or known in the art, such as phage display, as described inExample 6, below.

Additional anti-CD45 antibodies that can be used in conjunction with thepatient conditioning methods described herein include anti-CD45 antibodyT29/33, which is described, for instance, in Morikawa et al., Int. J.Hematol. 54:495-504, 1991, the disclosure of which is incorporatedherein by reference as it pertains to anti-CD45 antibodies.

The disclosures of each of the foregoing publications are incorporatedherein by reference as they pertain to anti-CD45 antibodies. Antibodiesand antigen-binding fragments that may be used in conjunction with thecompositions and methods described herein include the above-describedantibodies and antigen-binding fragments thereof, as well as humanizedvariants of those non-human antibodies and antigen-binding fragmentsdescribed above and antibodies or antigen-binding fragments that bindthe same epitope as those described above, as assessed, for instance, byway of a competitive CD45 binding assay.

Anti-CD135 Antibodies

The present invention is based in part on the discovery that antibodiesand antigen-binding fragments thereof capable of binding CD135 (alsoreferred to as Flk2 and Flt3) can be used as therapeutic agents to treatcancers and autoimmune diseases directly, as well as to promote theengraftment of transplanted hematopoietic stem cells in a patient inneed of transplant therapy. Additionally, it has been discovered thatligands that bind CD135, such as human Flt3 ligand, can be used to treatcancers and autoimmune diseases directly, as well as to promote theengraftment of transplanted hematopoietic stem cells in a patient inneed of transplant therapy. These ligands, such as human Flt3 ligand,can be covalently bound to an effector domain, such as an Fc domain, forinstance, in order to promote antibody-dependent cell-mediatedcytotoxicity (ADCC).

Hematopoietic stem cells have been shown to express CD135, as thisantigen is a receptor tyrosine kinase that has been reported to promotethe up-regulation of Mcl-1, which in turn modulates cell survivalthroughout hematopoiesis (see, e.g., Kikushige et al., J. Immunol.180:7358-7367, 2008, the disclosure of which is incorporated herein byreference as it pertains to the expression of CD135 by hematopoieticstem cells). Antibodies, antigen-binding fragments thereof, and ligandscapable of binding this cell-surface antigen can be identified usingtechniques known in the art and described herein, such as byimmunization, computational modeling techniques, and in vitro selectionmethods, such as the phage display and cell-based display platformsdescribed below.

Anti-CD135 antibodies that can be used in conjunction with the patientconditioning methods disclosed herein include those that have one ormore, or all, of the following CDRs:

(SEQ ID NO: 1) a. a CDR-H1 having the amino acid sequence SYYMH;(SEQ ID NO: 2) b. a CDR-H2 having the amino acid sequenceIINPSGGSTSYAQKFQG; (SEQ ID NO: 3)c. a CDR-H3 having the amino acid sequence GVGAHDAFDI or (SEQ ID NO: 4)VVAAAVADY; (SEQ ID NO: 5) d. a CDR-L1 having the amino acid sequenceRSSQSLLHSNGNNYLD or (SEQ ID NO: 6) RSSQSLLHSNGYNYLD; (SEQ ID NO: 7)e. a CDR-L2 having the amino acid sequence LGSNRAS; and (SEQ ID NO: 8)f. a CDR-L3 having the amino acid sequence MQGTHPAIS or (SEQ ID NO: 9)MQSLQTPFT.

Additional Anti-CD135 antibodies that can be used in conjunction withthe patient conditioning methods disclosed herein include those thathave one or more, or all, of the following CDRs:

(SEQ ID NO: 10) a. a CDR-H1 having the amino acid sequence SYAIS;(SEQ ID NO: 11) b. a CDR-H2 having the amino acid sequenceGIIPIFGTANYAQKFQG; (SEQ ID NO: 12)c. a CDR-H3 having the amino acid sequence FALFGFREQAFDI;(SEQ ID NO: 13) d. a CDR-L1 having the amino acid sequence RASQSISSYLN;(SEQ ID NO: 14) e. a CDR-L2 having the amino acid sequence AASSLQS; and(SEQ ID NO: 15) f. a CDR-L3 having the amino acid sequence QQSYSTPFT.

The foregoing antibodies are described, e.g., in U.S. Pat. No.8,071,099, the disclosure of which is incorporated herein by referenceas it pertains to anti-CD135 antibodies and antigen-binding fragmentsthereof. The antibodies and fragments thereof disclosed in U.S. Pat. No.8,071,099, such as IMC-EB10 and IMC-NC7, can be used in conjunction withthe methods disclosed herein.

In addition to the above, anti-CD135 antibodies that can be used inconjunction with the patient conditioning methods described hereininclude those that have one or more, or all, of the following CDRs:

(SEQ ID NO: 22) a. a CDR-H1 having the amino acid sequence SYWMH;(SEQ ID NO: 23) b. a CDR-H2 having the amino acid sequenceEIDPSDSYKDYNQKFK; (SEQ ID NO: 24)c. a CDR-H3 having the amino acid sequence AITTTPFDF; (SEQ ID NO: 25)d. a CDR-L1 having the amino acid sequence RASQSISNNLH; (SEQ ID NO: 26)e. a CDR-L2 having the amino acid sequence YASQSIS; and (SEQ ID NO: 27)f. a CDR-L3 having the amino acid sequence QQSNTWPYT.

Additional Anti-CD135 antibodies that can be used in conjunction withthe patient conditioning methods disclosed herein include those thathave one or more, or all, of the following CDRs:

(SEQ ID NO: 28) a. a CDR-H1 having the amino acid sequence NYGLH;(SEQ ID NO: 29) b. a CDR-H2 having the amino acid sequenceVIWSGGSTDYNAAFIS; (SEQ ID NO: 30)c. a CDR-H3 having the amino acid sequence KGGIYYANHYYAMDY;(SEQ ID NO: 31) d. a CDR-L1 having the amino acid sequenceKSSQSLLNSGNQKNYM; (SEQ ID NO: 32)e. a CDR-L2 having the amino acid sequence GASTRES; and (SEQ ID NO: 33)f. a CDR-L3 having the amino acid sequence QNDHSYPLT.

The foregoing antibodies are described, e.g., in U.S. Pat. No.9,023,996, the disclosure of which is incorporated herein by referenceas it pertains to anti-CD135 antibodies and antigen-binding fragmentsthereof. The antibodies and fragments thereof disclosed in U.S. Pat. No.9,023,996, such as clones 4G8 and BV10, can be used in conjunction withthe methods disclosed herein.

Other anti-CD135 antibodies that can be used in conjunction with thepatient conditioning methods described herein include, for instance,antibodies produced and released by American Type Culture Collection(ATCC) Accession No. ATCC HB 11,557, which is described, for example, inU.S. Pat. No. 5,635,388, the disclosure of which is incorporated hereinby reference as it pertains to anti-CD135 antibodies. Additionalanti-CD135 antibodies that can be used in conjunction with the patientconditioning methods described herein include, for instance, antibodiesproduced and released by hybridoma cells that are deposited at theInternational Depositary Authority DSMZ-Deutsche Sammlung vonMikroorganismen and Zellkulturen GmbH Mascheroder Weg 1 b, D-38124Braunschweig, Germany, as of Dec. 19, 1995 under No. DSM ACC2249 inaccordance with the Budapest Treaty, and designated 4G8B4B 12, which aredescribed, for example, in U.S. Pat. No. 6,156,882, the disclosure ofwhich is incorporated herein by reference as it pertains to anti-CD135antibodies. Additional anti-CD135 antibodies that can be used inconjunction with the patient conditioning methods described hereininclude, for instance, antibodies produced and released by hybridomacells that were deposited on Dec. 19, 1995 under No. DSM ACC2248 at theGerman Collection of Microorganisms and Cell Cultures Ltd. in accordancewith the Budapest Treaty, and designated BV10A4H2, which are described,for example, in U.S. Pat. No. 5,777,084, the disclosure of which isincorporated herein by reference as it pertains to anti-CD135antibodies. Additional anti-CD135 antibodies that can be used inconjunction with the patient-conditioning methods described hereininclude, for instance, antibodies produced and released by ATCCAccession No. FTA-4089, which is described, for example, in U.S. Pat.No. 7,183,385, the disclosure of which is incorporated herein byreference as it pertains to anti-CD135 antibodies. Additional anti-CD135antibodies are described in U.S. Pat. No. 5,548,065 (including, forinstance, anti-CD135 antibodies, antigen-binding fragments thereof, andligands produced and released by ATCC Accession Nos. CRL 10907, CRL10935, CRL 10936, and CRL 11005) and U.S. Pat. No. 9,109,227, as well asin US 2009/0054358, the disclosures of each of which are incorporatedherein by reference as they pertain to anti-CD135 antibodies. Otheranti-CD135 antibodies for use in conjunction with the methods describedherein include antibodies produced by the rabbit hybridoma cell lineC24D9, and humanized variants thereof, as described, for example, in US2010/0093008, the disclosure of which is incorporated herein byreference as it pertains to anti-CD135 antibodies.

In addition to the above, antibodies and antigen-binding fragmentsthereof capable of binding CD135 include those described in WO1995/007348, the disclosure of which is incorporated herein by referenceas it pertains to anti-CD135 antibodies. For instance, humanizedvariants of rat antibody 19A, produced and released by ATCC AccessionNo. HB 11442, humanized variants of antibody 23H, produced and releasedby ATCC Accession No. HB 11443, humanized variants of antibody 2A13,produced and released by ATCC Accession No. 11444, humanized variants ofmurine antibody 6J11, produced and released by ATCC Accession No. HB11445, and humanized variants of rat antibody 7 IE, as described in WO1995/007348, can be used in conjunction with the methods describedherein to condition a patient prior to hematopoietic stem celltransplant therapy.

In addition to antibodies and antigen-binding fragments, CD135 ligands,such as human Flt3 ligand, can be administered to a patient according tothe methods described herein, such as to treat a cancer it autoimmunedisorder or to condition a patient prior to hematopoietic stem celltransplant therapy. For instance, CD135 ligands, such as human Flt3ligand, can be conjugated to a cytotoxin (e.g., according to the methodsdescribed below or known in the art) or another effector molecule, suchas an Fc domain. CD135 ligands for use with the methods described hereininclude, for example, human Flt3 ligand-IgG1 Fc conjugates, human Flt3ligand-IgG2 Fc conjugates, human Flt3 ligand-IgG3 Fc conjugates, andhuman Flt3 ligand-IgG4 Fc conjugates, such as those produced byADIPOGEN® (San Diego, Calif.), product number AG-40B-0119.

The disclosures of each of the foregoing publications are incorporatedherein by reference as they pertain to anti-CD135 antibodies. Antibodiesand antigen-binding fragments that may be used in conjunction with thecompositions and methods described herein include the above-describedantibodies and antigen-binding fragments thereof, as well as humanizedvariants of those non-human antibodies and antigen-binding fragmentsdescribed above and antibodies or antigen-binding fragments that bindthe same epitope as those described above, as assessed, for instance, byway of a competitive CD135 binding assay.

Anti-CD34 Antibodies

Antibodies and antigen-binding fragments capable of binding human CD34(mRNA NCBI Reference Sequence: NM_001025109.1, Protein NCBI ReferenceSequence: NP_001020280.1) can be used in conjunction with thecompositions and methods described herein. An understanding of thepolymorphisms affecting the coding region or extracellular domain ofCD34 in a significant percentage of the population has not yet beenestablished. There are two isoforms of CD34 that differ in the length oftheir cytoplasmic tail (long and short). Recently, the long isoform wasused to generate a stable cell line expressing CD34 that could be usedas an immunogen (see, e.g., Adv. Pharm. Bull. 5:69-75, 2015). CD34, suchas the long isoform of CD34, can be used as an immunogen in order toidentify antibodies and antigen-binding fragments thereof capable ofbinding CD34 and suitable, for example, for the treatment of cancers andautoimmune diseases, as well as for use as a conditioning agent prior tohematopoietic stem cell transplant therapy.

CD34 antibodies that can be used in conjunction with the methodsdescribed herein include, without limitation, antibodies produced andreleased from ATCC Accession No. AC133.1 and HB 12346, as described, forexample, in U.S. Pat. No. 5,843,633.

The disclosure of the foregoing publication is incorporated herein byreference as it pertains to anti-CD34 antibodies. Antibodies andantigen-binding fragments that may be used in conjunction with thecompositions and methods described herein include the above-describedantibodies and antigen-binding fragments thereof, as well as humanizedvariants of those non-human antibodies and antigen-binding fragmentsdescribed above and antibodies or antigen-binding fragments that bindthe same epitope as those described above, as assessed, for instance, byway of a competitive CD34 binding assay.

Anti-CD90 Antibodies

Antibodies and antigen-binding fragments thereof capable of bindinghuman CD90 can be used in conjunction with the compositions and methodsdescribed herein. Hematopoietic stem cells have been shown to expressCD90, as Lin-CD34+CD38−CD90+CD45RA− fractions of human cord blood areenriched have been reported to contain hematopoietic stem cells (see,e.g., Majeti et al., Cell Stem Cell 1:635-645, 2007, the disclosure ofwhich is incorporated herein by reference as it pertains to theexpression of CD90 by hematopoietic stem cells). Antibodies andantigen-binding fragments capable of binding this cell-surface antigencan be raised using techniques known in the art and described herein,such as by immunization and in vitro selection techniques includingphage display and cell-based display platforms described below.

Anti-CD90 antibodies that can be used in conjunction with the methodsdescribed herein include, without limitation, EPR3132, EPR3133, AF-9,5E10, F15-42-1, 7E1B11, ab189367, aTHy-1A1, ab106934, and ab110477,which are commercially available from ABCAM® (Cambridge, Mass.), as wellas humanized variants thereof.

Antibodies and antigen-binding fragments that may be used in conjunctionwith the compositions and methods described herein include theabove-described antibodies and antigen-binding fragments thereof, aswell as humanized variants of those non-human antibodies andantigen-binding fragments described above and antibodies orantigen-binding fragments that bind the same epitope as those describedabove, as assessed, for instance, by way of a competitive CD90 bindingassay.

Anti-CD110 Antibodies

Antibodies and antigen-binding fragments thereof capable of bindinghuman CD110 (also referred to as c-mpl and thrombopoietic receptor(TPO)) can be used in conjunction with the compositions and methodsdescribed herein. Hematopoietic stem cells have been shown to expressCD110, and the expression of CD110 has been correlated withhematopoietic stem cell functional potential and decreases ashematopoietic stem cells differentiate and progress through thehematopoietic lineage (see, e.g., Ninos et al., J. Transl. Med. 4:9,2006, the disclosure of which is incorporated herein by reference as itpertains to the expression of CD110 by hematopoietic stem cells).Antibodies and antigen-binding fragments capable of binding thiscell-surface antigen can be raised using techniques known in the art anddescribed herein, such as by immunization and in vitro selectiontechniques including phage display and cell-based display platformsdescribed below.

Anti-CD110 antibodies that can be used in conjunction with the methodsdescribed herein, such as the cancer treatment methods, autoimmunedisease treatment methods, and patient conditioning methods describedherein include clones BAH-1, 1.78.1, and 1.6.1, as described, forexample, in De Gobbi et al., Epigenetics Chromatin 4:9, 2011;Erickson-Miller et al., BMC Cancer 12:405, 2012; and Petit Cocault etal., Exp. Hematol. 44:297-302, 2016, the disclosures of each of whichare incorporated herein by reference as they pertain to anti-CD110antibodies.

The disclosures of each of the foregoing publications are incorporatedherein by reference as they pertain to anti-CD110 antibodies. Antibodiesand antigen-binding fragments that may be used in conjunction with thecompositions and methods described herein include the above-describedantibodies and antigen-binding fragments thereof, as well as humanizedvariants of those non-human antibodies and antigen-binding fragmentsdescribed above and antibodies or antigen-binding fragments that bindthe same epitope as those described above, as assessed, for instance, byway of a competitive CD110 binding assay.

Additional Antibodies and Antigen-Binding Fragments Thereof

Antibodies and ligands for use in conjunction with the methods describedherein include variants of those antibodies described above, such asantibody fragments that contain or lack an Fc domain, as well ashumanized variants of non-human antibodies described herein andantibody-like protein scaffolds (e.g., ¹⁰Fn3 domains) containing one ormore, or all, of the CDRs or equivalent regions thereof of an antibody,antibody fragment, or ligand described herein. Exemplary antigen-bindingfragments of the foregoing antibodies include a dual-variableimmunoglobulin domain, a single-chain Fv molecule (scFv), a diabody, atriabody, a nanobody, an antibody-like protein scaffold, a Fv fragment,a Fab fragment, a F(ab′)₂ molecule, and a tandem di-scFv, among others.

Methods of Combination Therapy

Using the methods described herein, a physician of skill in the art maytreat a patient for cancer or an autoimmune disease or may condition apatient prior to hematopoietic stem cell transplant therapy byadministering one or more antibodies, antigen-binding fragments thereof,or ligands described herein. For instance, a physician may treat acancer or autoimmune disease or condition a patient for hematopoieticstem cell transplant therapy by administering an antibody,antigen-binding fragment thereof, or ligand that recognizes and bindsCD45 (e.g., CD45RO) alone or in combination with an antibody,antigen-binding fragment thereof, or ligand that recognizes and bindsCD135, an antibody, antigen-binding fragment thereof, or ligand thatrecognizes and binds CD34, an antibody, antigen-binding fragmentthereof, or ligand that recognizes and binds CD90, and/or an antibody,antigen-binding fragment thereof, or ligand that recognizes and bindsCD110. Dosing regimens for combination therapy may include sequentialadministration of two or more of the above antibodies, antigen-bindingfragments thereof, or ligands, such that a first antibody,antigen-binding fragment thereof, or ligand is administered to a patientat a designated time point, and a second antibody, antigen-bindingfragment thereof, or ligand is subsequently administered to a patient ata later time point, for example, so as to optimize the pharmacokineticprofile of the combined therapeutic dosage. Two or more antibodies,antigen-binding fragments thereof, or ligands that each bind one or moreof the hematopoietic stem cell antigens described herein can beadministered to a patient (e.g., a human patient) concurrently, may beadmixed with one another in a single pharmaceutical composition, or maybe administered at different times (e.g., within 1-24 hours of oneanother, such as within 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20hours, 21 hours, 22 hours, 23 hours, 24 hours, or more, of one another).

Antibodies, antigen-binding fragments thereof, and ligands can beadministered to a patient so as to recognize and bind multiplehematopoietic stem cell antigens using a single antibody. This can beachieved, for example, using bi-specific and multi-specific antibodies.For instance, bi-specific antibodies containing an antigen-bindingfragment that selectively binds CD45 (e.g., CD45RO) and anotherantigen-binding fragment that selectively binds CD135 can beadministered to a patient in need of hematopoietic stem cell transplanttherapy in order to deplete CD45+ CD135+ cells, thereby creating avacancy in hematopoietic tissue (e.g., the bone marrow) that can befilled by an exogenous hematopoietic stem cell transplant. Similarly,bi-specific antibodies containing an antigen-binding fragment thatselectively binds CD45 (e.g., CD45RO) and CD34 can be administered to apatient in order to deplete CD34+ CD45+ cells prior to hematopoieticstem cell transplant therapy. Bi-specific antibodies containing anantigen-binding fragment that selectively binds CD45 (e.g., CD45RO) andCD90 can be similarly administered to a patient, such as a patientsuffering from cancer or an autoimmune disease, or in order to depleteCD45+CD90+ cells prior to hematopoietic stem cell transplant therapy.Additionally, bi-specific antibodies containing an antigen-bindingfragment that selectively binds CD45 (e.g., CD45RO) and CD110 can beadministered to a patient, for instance, in order to deplete CD45+CD110+ cells prior to hematopoietic stem cell transplantation.

In some embodiments, bi-specific antibodies containing anantigen-binding fragment that selectively binds CD135 and anotherantigen-binding fragment that selectively binds CD34 can be administeredto a patient, such as a patient suffering from cancer, an autoimmunedisease, or in need of hematopoietic stem cell transplant therapy inorder to deplete CD34+ CD135+ cells, thereby creating a vacancy inhematopoietic tissue (e.g., the bone marrow) that can be filled by anexogenous hematopoietic stem cell transplant. Similarly, bi-specificantibodies containing an antigen-binding fragment that selectively bindsCD135 and CD90 can be administered to a patient in order to depleteCD90+ CD135+ cells prior to hematopoietic stem cell transplant therapy.Bi-specific antibodies containing an antigen-binding fragment thatselectively binds CD135 and CD110 can be similarly administered to apatient, for instance, in order to deplete CD110+ CD135+ cells prior tohematopoietic stem cell transplant therapy.

In some embodiments, bi-specific antibodies containing anantigen-binding fragment that selectively binds CD34 and anotherantigen-binding fragment that selectively binds CD90 can be administeredto a patient suffering from cancer, an autoimmune disease, or in need ofhematopoietic stem cell transplant therapy in order to deplete CD34+CD90+ cells, thereby creating a vacancy in hematopoietic tissue (e.g.,the bone marrow) that can be filled by an exogenous hematopoietic stemcell transplant. Similarly, bi-specific antibodies containing anantigen-binding fragment that selectively binds CD34 and CD110 can beadministered to a patient, for instance, in order to deplete CD34+CD110+ cells prior to hematopoietic stem cell transplant therapy.

In some embodiments, bi-specific antibodies containing anantigen-binding fragment that selectively binds CD90 and anotherantigen-binding fragment that selectively binds CD110 can beadministered to a patient suffering from cancer, an autoimmune disease,or in need of hematopoietic stem cell transplant therapy in order todeplete CD90+ CD110+ cells, thereby creating a vacancy in hematopoietictissue (e.g., the bone marrow) that can be filled by an exogenoushematopoietic stem cell transplant.

Methods of Identifying Antibodies and Ligands

Methods for high throughput screening of antibody, antibody fragment,and ligand libraries for molecules capable of binding CD45 (e.g.,CD45RO), CD135, CD34, CD90, and CD110 can be used to identify andaffinity mature antibodies useful for treating cancers, autoimmunediseases, and conditioning a patient (e.g., a human patient) in need ofhematopoietic stem cell therapy as described herein. Such methodsinclude in vitro display techniques known in the art, such as phagedisplay, bacterial display, yeast display, mammalian cell display,ribosome display, mRNA display, and cDNA display, among others. The useof phage display to isolate ligands that bind biologically relevantmolecules has been reviewed, for example, in Felici et al., Biotechnol.Annual Rev. 1:149-183, 1995; Katz, Annual Rev. Biophys. Biomol. Struct.26:27-45, 1997; and Hoogenboom et al., Immunotechnology 4:1-20, 1998,the disclosures of each of which are incorporated herein by reference asthey pertain to in vitro display techniques. Randomized combinatorialpeptide libraries have been constructed to select for polypeptides thatbind cell surface antigens as described in Kay, Perspect. Drug DiscoveryDes. 2:251-268, 1995 and Kay et al., Mol. Divers. 1:139-140, 1996, thedisclosures of each of which are incorporated herein by reference asthey pertain to the discovery of antigen-binding molecules. Proteins,such as multimeric proteins, have been successfully phage-displayed asfunctional molecules (see, for example, EP 0349578; EP 4527839; and EP0589877, as well as Chiswell and McCafferty, Trends Biotechnol. 10:80-841992, the disclosures of each of which are incorporated herein byreference as they pertain to the use of in vitro display techniques forthe discovery of antigen-binding molecules). In addition, functionalantibody fragments, such as Fab and scFv fragments, have been expressedin in vitro display formats (see, for example, McCafferty et al., Nature348:552-554, 1990; Barbas et al., Proc. Natl. Acad. Sci. USA88:7978-7982, 1991; and Clackson et al., Nature 352:624-628, 1991, thedisclosures of each of which are incorporated herein by reference asthey pertain to in vitro display platforms for the discovery ofantigen-binding molecules). These techniques, among others, can be usedto identify and improve the affinity of antibodies that bindhematopoietic stem cell antigens (such as CD45 (e.g., CD45RO), CD135,CD34, CD90, and CD110) that can in turn be used to deplete endogenoushematopoietic stem cells in a patient (e.g., a human patient) in need ofhematopoietic stem cell transplant therapy.

In addition to in vitro display techniques, computational modelingtechniques can be used to design and identify antibodies, antibodyfragments, and ligands in silico that bind CD45 (e.g., CD45RO), CD135,CD34, CD90, or CD110. For example, using computational modelingtechniques, one of skill in the art can screen libraries of antibodies,antibody fragments, and ligands in silico for molecules capable ofbinding specific epitopes on CD45 (e.g., CD45RO), CD135, CD34, CD90, andCD110, such as extracellular epitopes of these antigens. The antibodies,antigen-binding fragments thereof, and ligands identified by thesecomputational techniques can be used in conjunction with the therapeuticmethods described herein, such as the cancer and autoimmune diseasetreatment methods described herein and the patient conditioningprocedures described herein.

Additional techniques can be used to identify antibodies,antigen-binding fragments thereof, and ligands that bind CD45 (e.g.,CD45RO), CD135, CD34, CD90, and/or CD110 on the surface of a cell (e.g.,a cancer cell, autoimmune cell, or a hematopoietic stem cell) and thatare internalized by the cell, for instance, by receptor-mediatedendocytosis. For example, the in vitro display techniques describedabove can be adapted to screen for antibodies, antigen-binding fragmentsthereof, and ligands that bind CD45 (e.g., CD45RO), CD135, CD34, CD90,and/or CD110 on the surface of a cancer cell, autoimmune cell, orhematopoietic stem cell and that are subsequently internalized. Phagedisplay represents one such technique that can be used in conjunctionwith this screening paradigm. To identify antibodies, fragments thereof,and ligands that bind one or more of the above targets and aresubsequently internalized by cancer cells, autoimmune cells, orhematopoietic stem cells, one of skill in the art can adapt the phagedisplay techniques described, for example, in Williams et al., Leukemia19:1432-1438, 2005, the disclosure of which is incorporated herein byreference in its entirety. For example, using mutagenesis methods knownin the art, recombinant phage libraries can be produced that encodeantibodies, antibody fragments, such as scFv fragments, Fab fragments,diabodies, triabodies, and ¹⁰Fn3 domains, among others, or ligands thatcontain randomized amino acid cassettes (e.g., in one or more, or all,of the CDRs or equivalent regions thereof or an antibody or antibodyfragment). The framework regions, hinge, Fc domain, and other regions ofthe antibodies or antibody fragments may be designed such that they arenon-immunogenic in humans, for instance, by virtue of having humangermline antibody sequences or sequences that exhibit only minorvariations relative to human germline antibodies.

Using phage display techniques described herein or known in the art,phage libraries containing randomized antibodies, antibody fragments, orligands covalently bound to the phage particles can be incubated withCD45 (e.g., CD45RO), CD135, CD34, CD90, and/or CD110 antigen, forinstance, by first incubating the phage library with blocking agents(such as, for instance, milk protein, bovine serum albumin, and/or IgGso as to remove phage encoding antibodies, fragments thereof, or ligandsthat exhibit non-specific protein binding and phage that encodeantibodies or fragments thereof that bind Fc domains, and thenincubating the phage library with a population of hematopoietic stemcells. The phage library can be incubated with the target cells, such ascancer cells, autoimmune cells, or hematopoietic stem cells for a timesufficient to allow CD45-specific, CD135-specific, CD34-specific,CD90-specific, and/or CD110-specific antibodies, antigen-bindingfragments thereof, or ligands to bind cell-surface antigen and tosubsequently be internalized by the cancer cells, autoimmune cells, orhematopoietic stem cells (e.g., from 30 minutes to 6 hours at 4° C.,such as 1 hour at 4° C.). Phage containing antibodies, fragmentsthereof, or ligands that do not exhibit sufficient affinity for one ormore of these antigens so as to permit binding to, and internalizationby, cancer cells, autoimmune cells, or hematopoietic stem cells cansubsequently be removed by washing the cells, for instance, with cold(4° C.) 0.1 M glycine buffer at pH 2.8. Phage bound to antibodies,fragments thereof, or ligands that have been internalized by the cancercells, autoimmune cells, or hematopoietic stem cells can be identified,for instance, by lysing the cells and recovering internalized phage fromthe cell culture medium. The phage can then be amplified in bacterialcells, for example, by incubating bacterial cells with recovered phagein 2×YT medium using methods known in the art. Phage recovered from thismedium can then be characterized, for instance, by determining thenucleic acid sequence of the gene(s) encoding the antibodies, fragmentsthereof, or ligands inserted within the phage genome. The encodedantibodies, fragments thereof, or ligands can subsequently be preparedde novo by chemical synthesis (for instance, of antibody fragments, suchas scFv fragments, or ligands) or by recombinant expression (forinstance, of full-length antibodies).

The internalizing capacity of the prepared antibodies, fragmentsthereof, or ligands can be assessed, for instance, using radionuclideinternalization assays known in the art. For example, antibodies,fragments thereof, or ligands identified using in vitro displaytechniques described herein or known in the art can be functionalized byincorporation of a radioactive isotope, such as ¹⁸F, ⁷⁵Br, ⁷⁷Br, ¹²²I,¹²³I, ¹²⁴I, ¹²⁵I, ¹²⁹I, ¹³¹I, ²¹¹At, ⁶⁷Ga, ¹¹¹In, ⁹⁹Tc, ¹⁶⁹Yb, ¹⁸⁶Re,⁶⁴Cu, ⁶⁷Cu, ¹⁷⁷Lu, ⁷⁷As, ⁷²As, ⁸⁶Y, ⁹⁰Y, ⁸⁹Zr, ²¹²Bi, ²¹³Bi, or ²²⁵Ac.For instance, radioactive halogens, such as ¹⁸F, ⁷⁵Br, ⁷⁷Br, ¹²²I, ¹²³I,¹²⁴I, ¹²⁵I, ¹²⁹I, ¹³¹I, ²¹¹At, can be incorporated into antibodies,fragments thereof, or ligands using beads, such as polystyrene beads,containing electrophilic halogen reagents (e.g., Iodination Beads,Thermo Fisher Scientific, Inc., Cambridge, Mass.). Radiolabeledantibodies, fragments thereof, or ligands can be incubated with cancercells, autoimmune cells, or hematopoietic stem cells for a timesufficient to permit internalization (e.g., from 30 minutes to 6 hoursat 4° C., such as 1 hour at 4° C.). The cells can then be washed toremove non-internalized antibodies, fragments thereof, or ligands (e.g.,using cold (4° C.) 0.1 M glycine buffer at pH 2.8). Internalizedantibodies, fragments thereof, or ligands can be identified by detectingthe emitted radiation (e.g., γ-radiation) of the resulting cancer cells,autoimmune cells, or hematopoietic stem cells in comparison with theemitted radiation (e.g., γ-radiation) of the recovered wash buffer.

Drug-Antibody Conjugates and Drug-Ligand Conjugates Cytotoxins

Antibodies, antigen-binding fragments thereof, and ligands describedherein (e.g., antibodies, antigen-binding fragments, and ligands thatrecognize and bind CD45 (such as CD45RO), CD135, CD34, CD90, and/orCD110) can be conjugated to a cytotoxin, such as pseudomonas exotoxin A,deBouganin, diphtheria toxin, an amatoxin, such as α-amanitin, saporin,maytansine, a maytansinoid, an auristatin, an anthracycline, acalicheamicin, irinotecan, SN-38, a duocarmycin, apyrrolobenzodiazepine, a pyrrolobenzodiazepine dimer, anindolinobenzodiazepine, and an indolinobenzodiazepine dimer, or avariant thereof, or another cytotoxic compound described herein or knownin the art, for example, in order to treat a cancer or autoimmunedisease described herein or to promote the depletion of endogenoushematopoietic stem cells upon administration to a patient (e.g., a humanpatient) in need of hematopoietic stem cell transplant therapy. In someembodiments, the cytotoxic molecule is conjugated to an internalizingantibody, antigen-binding fragment thereof, or ligand, such thatfollowing the cellular uptake of the antibody, antigen-binding fragment,or ligand, the cytotoxin may access its intracellular target and mediateendogenous hematopoietic cell death. Cytotoxins suitable for use withthe compositions and methods described herein include DNA-intercalatingagents, (e.g., anthracyclines), agents capable of disrupting the mitoticspindle apparatus (e.g., vinca alkaloids, maytansine, maytansinoids, andderivatives thereof), RNA polymerase inhibitors (e.g., an amatoxin, suchas α-amanitin and derivatives thereof), agents capable of disruptingprotein biosynthesis (e.g., agents that exhibit rRNA N-glycosidaseactivity, such as saporin and ricin A-chain), among others known in theart.

In some embodiments, the cytotoxin is an amatoxin or derivative thereof,such as α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanin,amaninamide, amanullin, amanullinic acid, and proamanullin. Forinstance, the antibodies, antigen-binding fragments, and ligandsdescribed herein may be bound to an amatoxin so as to form a conjugaterepresented by the formula Ab-Am, wherein Ab is the antibody,antigen-binding fragment thereof, or ligand, and Am is an amatoxin. Insome embodiments, Am is represented by formula (I)

wherein R₁ is H, OH, OR_(A), or OR_(C);

R₂ is H, OH, OR_(B), or OR_(C);

R_(A) and R_(B), together with the oxygen atoms to which they are bound,combine to form an optionally substituted 5-membered heterocyclolalkylgroup;

R₃ is H, R_(C), or R_(D);

R₄ is H, OH, OR_(C), OR_(D), R_(C), or R_(D);

R₅ is H, OH, OR_(C), OR_(D), R_(C), or R_(D);

R₆ is H, OH, OR_(C), OR_(D), R_(C), or R_(D);

R₇ is H, OH, OR_(C), OR_(D), R_(C), or R_(D);

R₈ is OH, NH₂, OR_(C), OR_(D), NHR_(C), or NR_(C)R_(D);

R₉ is H, OH, OR_(C), or OR_(D);

X is —S—, —S(O)—, or —SO₂—;

R_(C) is -L-Z;

R_(D) is optionally substituted alkyl (e.g., C₁-C₆ alkyl), optionallysubstituted heteroalkyl (e.g., C₁-C₆ heteroalkyl), optionallysubstituted alkenyl (e.g., C₂-C₆ alkenyl), optionally substitutedheteroalkenyl (e.g., C₂-C₆ heteroalkenyl), optionally substitutedalkynyl (e.g., C₂-C₆ alkynyl), optionally substituted heteroalkynyl(e.g., C₂-C₆ heteroalkynyl), optionally substituted cycloalkyl,optionally substituted heterocycloalkyl, optionally substituted aryl, oroptionally substituted heteroaryl;

L is a linker, such as optionally substituted alkylene (e.g., C₁-C₆alkylene), optionally substituted heteroalkylene (C₁-C₆ heteroalkylene),optionally substituted alkenylene (e.g., C₂-C₆ alkenylene), optionallysubstituted heteroalkenylene (e.g., C₂-C₆ heteroalkenylene), optionallysubstituted alkynylene (e.g., C₂-C₆ alkynylene), optionally substitutedheteroalkynylene (e.g., C₂-C₆ heteroalkynylene), optionally substitutedcycloalkylene, optionally substituted heterocycloalkylene, optionallysubstituted arylene, or optionally substituted heteroarylene; and

Z is a chemical moiety formed from a coupling reaction between areactive substituent present on L and a reactive substituent presentwithin an antibody, antigen-binding fragment thereof, or ligand thatbinds CD45 (such as CD45RO), CD135, CD34, CD90, and/or CD110.

In some embodiments, Am contains exactly one R_(C) substituent.

In some embodiments, Am is represented by formula (IA)

wherein R₁ is H, OH, OR_(A), or OR_(C);

R₂ is H, OH, OR_(B), or OR_(C);

R_(A) and R_(B), together with the oxygen atoms to which they are bound,combine to form an optionally substituted 5-membered heterocyclolalkylgroup;

R₃ is H, R_(C), or R_(D);

R₄ is H, OH, OR_(C), OR_(B), R_(C), or R_(D);

R₅ is H, OH, OR_(C), OR_(B), R_(C), or R_(D);

R₆ is H, OH, OR_(C), OR_(B), R_(C), or R_(D);

R₇ is H, OH, OR_(C), OR_(B), R_(C), or R_(D);

R₈ is OH, NH₂, OR_(C), OR_(B), NHR_(C), or NR_(C)R_(D);

R₉ is H, OH, OR_(C), or OR_(D);

X is —S—, —S(O)—, or —SO₂—;

R_(C) is -L-Z;

R_(D) is optionally substituted alkyl (e.g., C₁-C₆ alkyl), optionallysubstituted heteroalkyl (e.g., C₁-C₆ heteroalkyl), optionallysubstituted alkenyl (e.g., C₂-C₆ alkenyl), optionally substitutedheteroalkenyl (e.g., C₂-C₆ heteroalkenyl), optionally substitutedalkynyl (e.g., C₂-C₆ alkynyl), optionally substituted heteroalkynyl(e.g., C₂-C₆ heteroalkynyl), optionally substituted cycloalkyl,optionally substituted heterocycloalkyl, optionally substituted aryl, oroptionally substituted heteroaryl;

L is a linker, such as optionally substituted alkylene (e.g., C₁-C₆alkylene), optionally substituted heteroalkylene (C₁-C₆ heteroalkylene),optionally substituted alkenylene (e.g., C₂-C₆ alkenylene), optionallysubstituted heteroalkenylene (e.g., C₂-C₆ heteroalkenylene), optionallysubstituted alkynylene (e.g., C₂-C₆ alkynylene), optionally substitutedheteroalkynylene (e.g., C₂-C₆ heteroalkynylene), optionally substitutedcycloalkylene, optionally substituted heterocycloalkylene, optionallysubstituted arylene, or optionally substituted heteroarylene;

Z is a chemical moiety formed from a coupling reaction between areactive substituent present on L and a reactive substituent presentwithin an antibody, antigen-binding fragment thereof, or ligand thatbinds CD45 (such as CD45RO), CD135, CD34, CD90, and/or CD110; and

wherein Am contains exactly one R_(C) substituent.

In some embodiments, Am is represented by formula (IB)

wherein R₁ is H, OH, OR_(A), or OR_(C);

R₂ is H, OH, OR_(B), or OR_(C);

R_(A) and R_(B), together with the oxygen atoms to which they are bound,combine to form an optionally substituted 5-membered heterocyclolalkylgroup;

R₃ is H, R_(C), or R_(D);

R₄ is H, OH, OR_(C), OR_(D), R_(C), or R_(D);

R₅ is H, OH, OR_(C), OR_(D), R_(C), or R_(D);

R₆ is H, OH, OR_(C), OR_(D), R_(C), or R_(D);

R₇ is H, OH, OR_(C), OR_(D), R_(C), or R_(D);

R₈ is OH, NH₂, OR_(C), OR_(D), NHR_(C), or NR_(C)R_(D);

R₉ is H, OH, OR_(C), or OR_(D);

X is —S—, —S(O)—, or —SO₂—;

R_(C) is -L-Z;

R_(D) is optionally substituted alkyl (e.g., C₁-C₆ alkyl), optionallysubstituted heteroalkyl (e.g., C₁-C₆ heteroalkyl), optionallysubstituted alkenyl (e.g., C₂-C₆ alkenyl), optionally substitutedheteroalkenyl (e.g., C₂-C₆ heteroalkenyl), optionally substitutedalkynyl (e.g., C₂-C₆ alkynyl), optionally substituted heteroalkynyl(e.g., C₂-C₆ heteroalkynyl), optionally substituted cycloalkyl,optionally substituted heterocycloalkyl, optionally substituted aryl, oroptionally substituted heteroaryl;

L is a linker, such as optionally substituted alkylene (e.g., C₁-C₆alkylene), optionally substituted heteroalkylene (C₁-C₆ heteroalkylene),optionally substituted alkenylene (e.g., C₂-C₆ alkenylene), optionallysubstituted heteroalkenylene (e.g., C₂-C₆ heteroalkenylene), optionallysubstituted alkynylene (e.g., C₂-C₆ alkynylene), optionally substitutedheteroalkynylene (e.g., C₂-C₆ heteroalkynylene), optionally substitutedcycloalkylene, optionally substituted heterocycloalkylene, optionallysubstituted arylene, or optionally substituted heteroarylene;

Z is a chemical moiety formed from a coupling reaction between areactive substituent present on L and a reactive substituent presentwithin an antibody, antigen-binding fragment thereof, or ligand thatbinds CD45 (such as CD45RO), CD135, CD34, CD90, and/or CD110; and

wherein Am contains exactly one R_(C) substituent.

In some embodiments, R_(A) and R_(B), together with the oxygen atoms towhich they are bound, combine to form:

wherein Y is selected from O, S, NR_(E), and CR_(E)R_(E′), and

R_(E) and R_(E′) are each independently optionally substituted C₁-C₆alkylene-R_(C), optionally substituted C₁-C₆ heteroalkylene-R_(C),optionally substituted C₂-C₆ alkenylene-R_(C), optionally substitutedC₂-C₆ heteroalkenylene-R_(C), optionally substituted C₂-C₆alkynylene-R_(C), optionally substituted C₂-C₆ heteroalkynylene-R_(C),optionally substituted cycloalkylene-R_(C), optionally substitutedheterocycloalkylene-R_(C), optionally substituted arylene-R_(C), oroptionally substituted heteroarylene-R_(C).

In some embodiments, Am is represented by formula (IA) or formula (IB),

wherein R₁ is H, OH, OR_(A), or OR_(C);

R₂ is H, OH, OR_(B), or OR_(C);

R_(A) and R_(B), together with the oxygen atoms to which they are bound,combine to form:

R₃ is H or R_(C);

R₄ is H, OH, OR_(C), OR_(D), R_(C), or R_(D);

R₅ is H, OH, OR_(C), OR_(D), R_(C), or R_(D);

R₆ is H, OH, OR_(C), OR_(D), R_(C), or R_(D);

R₇ is H, OH, OR_(C), OR_(D), R_(C), or R_(D);

R₈ is OH, NH₂, OR_(C), or NHR_(C);

R₉ is H or OH; and

wherein R_(C) and R_(D) are each as defined above.

In some embodiments, Am is represented by formula (IA) or formula (IB),

wherein R₁ is H, OH, OR_(A), or OR_(C);

R₂ is H, OH, OR_(B), or OR_(C);

R_(A) and R_(B), together with the oxygen atoms to which they are bound,combine to form:

R₃ is H or R_(C);

R₄ and R₅ are each independently H, OH, OR_(C), R_(C), or OR_(D);

R₆ and R₇ are each H;

R₈ is OH, NH₂, OR_(C), or NHR_(C);

R₉ is H or OH; and

wherein R_(C) is as defined above.

In some embodiments, Am is represented by formula (IA) or formula (IB),

wherein R₁ is H, OH, or OR_(A);

R₂ is H, OH, or OR_(B);

R_(A) and R_(B), together with the oxygen atoms to which they are bound,combine to form:

R₃, R₄, R₆, and R₇ are each H;

R₅ is OR_(C);

R₈ is OH or NH₂;

R₉ is H or OH; and

wherein R_(C) is as defined above. Such amatoxin conjugates aredescribed, for example, in US Patent Application Publication No.2016/0002298, the disclosure of which is incorporated herein byreference in its entirety.

In some embodiments, Am is represented by formula (IA) or formula (IB),

wherein R₁ and R₂ are each independently H or OH;

R₃ is R_(C);

R₄, R₆, and R₇ are each H;

R₅ is H, OH, or OC₁-C₆ alkyl;

R₈ is OH or NH₂;

R₉ is H or OH; and

wherein R_(C) is as defined above. Such amatoxin conjugates aredescribed, for example, in US Patent Application Publication No.2014/0294865, the disclosure of which is incorporated herein byreference in its entirety.

In some embodiments, Am is represented by formula (IA) or formula (IB),

wherein R₁ and R₂ are each independently H or OH;

R₃, R₆, and R₇ are each H;

R₄ and R₅ are each independently H, OH, OR_(C), or R_(C);

R₈ is OH or NH₂;

R₉ is H or OH; and

wherein R_(C) is as defined above. Such amatoxin conjugates aredescribed, for example, in US Patent Application Publication No.2015/0218220, the disclosure of which is incorporated herein byreference in its entirety.

In some embodiments, Am is represented by formula (IA) or formula (IB),

wherein R₁ and R₂ are each independently H or OH;

R₃, R₆, and R₇ are each H;

R₄ and R₅ are each independently H or OH;

R₈ is OH, NH₂, OR_(C), or NHR_(C);

R₉ is H or OH; and

wherein R_(C) is as defined above. Such amatoxin conjugates aredescribed, for example, in U.S. Pat. Nos. 9,233,173 and 9,399,681, aswell as in US 2016/0089450, the disclosures of each of which areincorporated herein by reference in their entirety.

Additional amatoxins that may be used for conjugation to an antibody,antigen-binding fragment thereof, or ligand in accordance with thecompositions and methods described herein are described, for example, inWO 2016/142049; WO 2016/071856; and WO 2017/046658, the disclosures ofeach of which are incorporated herein by reference in their entirety.

In some embodiments, Am is represented by formula (II),

wherein X is S, SO, or SO₂;

R₁ is H or a linker covalently bound to the antibody or antigen-bindingfragment thereof;

R₂ is H or a linker covalently bound to the antibody or antigen-bindingfragment thereof; and

wherein when R₁ is H, R₂ is the linker, and when R₂ is H, R₁ is thelinker.

Antibodies, antigen-binding fragments, and ligands for use with thecompositions and methods described herein can be conjugated to anamatoxin, such as α-amanitin or a variant thereof, using conjugationtechniques known in the art or described herein. For instance,antibodies, antigen-binding fragments thereof, and ligands thatrecognize and bind CD45 (such as CD45RO), CD135, CD34, CD90, and/orCD110 can be conjugated to an amatoxin, such as α-amanitin or a variantthereof, as described in US 2015/0218220, the disclosure of which isincorporated herein by reference as it pertains, for example, toamatoxins, such as α-amanitin and variants thereof, as well as covalentlinkers that can be used for covalent conjugation.

Exemplary antibody-drug and ligand-drug conjugates useful in conjunctionwith the methods described herein may be formed by the reaction of anantibody, antigen-binding fragment thereof, or ligand with an amatoxinthat is conjugated to a linker containing a substituent suitable forreaction with a reactive residue on the antibody, antigen-bindingfragment thereof, or ligand. Amatoxins that are conjugated to a linkercontaining a substituent suitable for reaction with a reactive residueon the antibody, antigen-binding fragment thereof, or ligand describedherein include, without limitation,7′C-(4-(6-(maleimido)hexanoyl)piperazin-1-yl)-amatoxin;7′C-(4-(6-(maleimido)hexanamido)piperidin-1-yl)-amatoxin;7′C-(4-(6-(6-(maleimido)hexanamido)hexanoyl)piperazin-1-yl)-amatoxin;7′C-(4-(4-((maleimido)methyl)cyclohexanecarbonyl)piperazin-1-yl)-amatoxin;7′C-(4-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanoyl)piperazin-1-yl)-amatoxin;7′C-(4-(2-(6-(maleimido)hexanamido)ethyl)piperidin-1-yl)-amatoxin;7′C-(4-(2-(6-(6-(maleimido)hexanamido)hexanamido)ethyl)piperidin-1-yl)-amatoxin;7′C-(4-(2-(4-((maleimido)methyl)cyclohexanecarboxamido)ethyl)piperidin-1-yl)-amatoxin;7′C-(4-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)ethyl)piperidin-1-yl)-amatoxin;7′C-(4-(2-(3-carboxypropanamido)ethyl)piperidin-1-yl)-amatoxin;7′C-(4-(2-(2-bromoacetamido)ethyl)piperidin-1-yl)-amatoxin;7′C-(4-(2-(3-(pyridin-2-yldisulfanyl)propanamido)ethyl)piperidin-1-yl)-amatoxin;7′C-(4-(2-(4-(maleimido)butanamido)ethyl)piperidin-1-yl)-amatoxin;7′C-(4-(2-(maleimido)acetyl)piperazin-1-yl)-amatoxin;7′C-(4-(3-(maleimido)propanoyl)piperazin-1-yl)-amatoxin;7′C-(4-(4-(maleimido)butanoyl)piperazin-1-yl)-amatoxin;7′C-(4-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)ethyl)piperidin-1-yl)-amatoxin;7′C-(3-((6-(maleimido)hexanamido)methyl)pyrrolidin-1-yl)-amatoxin;7′C-(3-((6-(6-(maleimido)hexanamido)hexanamido)methyl)pyrrolidin-1-yl)-amatoxin;7′C-(3-((4-((maleimido)methyl)cyclohexanecarboxamido)methyl)pyrrolidin-1-yl)-amatoxin;7′C-(3-((6-((4-(maleimido)methyl)cyclohexanecarboxamido)hexanamido)methyl)pyrrolidin-1-yl)-amatoxin;7′C-(4-(2-(6-(2-(aminooxy)acetamido)hexanamido)ethyl)piperidin-1-yl)-amatoxin;7′C-(4-(2-(4-(2-(aminooxy)acetamido)butanamido)ethyl)piperidin-1-yl)-amatoxin;7′C-(4-(4-(2-(aminooxy)acetamido)butanoyl)piperazin-1-yl)-amatoxin;7′C-(4-(6-(2-(aminooxy)acetamido)hexanoyl)piperazin-1-yl)-amatoxin;7′C-((4-(6-(maleimido)hexanamido)piperidin-1-yl)methyl)-amatoxin;7′C-((4-(2-(6-(maleimido)hexanamido)ethyl)piperidin-1-yl)methyl)-amatoxin;7′C-((4-(6-(maleimido)hexanoyl)piperazin-1-yl)methyl)-amatoxin;(R)-7′C-((3-((6-(maleimido)hexanamido)methyl)pyrrolidin-1-yl)methyl)-amatoxin;(S)-7′C-((3-((6-(maleimido)hexanamido)methyl)pyrrolidin-1-yl)methyl)-amatoxin;7′C-((4-(2-(6-(6-(maleimido)hexanamido)hexanamido)ethyl)piperidin-1-yl)methyl)-amatoxin;7′C-((4-(2-(4-((maleimido)methyl)cyclohexanecarboxamido)ethyl)piperidin-1-yl)methyl)-amatoxin;7′C-((4-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)ethyl)piperidin-1-yl)methyl)-amatoxin;7′C-((4-(2-(6-(maleimido)hexanamido)ethyl)piperazin-1-yl)methyl)-amatoxin;7′C-((4-(2-(6-(6-(maleimido)hexanamido)hexanamido)ethyl)piperazin-1-yl)methyl)-amatoxin;7′C-((4-(2-(4-((maleimido)methyl)cyclohexanecarboxamido)ethyl)piperazin-1-yl)methyl)-amatoxin;7′C-((4-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)ethyl)piperazin-1-yl)methyl)-amatoxin;7′C-((3-((6-(6-(maleimido)hexanamido)hexanamido)-S-methyl)pyrrolidin-1-yl)methyl)-amatoxin;7′C-((3-((6-(6-(maleimido)hexanamido)hexanamido)-R-methyl)pyrrolidin-1-yl)methyl)-amatoxin;7′C-((3-((4-((maleimido)methyl)cyclohexanecarboxamido)-S-methyl)pyrrolidin-1-yl)methyl)-amatoxin;7′C-((3-((4-((maleimido)methyl)cyclohexanecarboxamido)-R-methyl)pyrrolidin-1-yl)methyl)-amatoxin;7′C-((3-((6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)methyl)pyrrolidin-1-yl)methyl)-amatoxin;7′C-((4-(2-(3-carboxypropanamido)ethyl)piperazin-1-yl)methyl)-amatoxin;7′C-((4-(6-(6-(maleimido)hexanamido)hexanoyl)piperazin-1-yl)methyl)-amatoxin;7′C-((4-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanoyl)piperazin-1-yl)methyl)-amatoxin;7′C-((4-(2-(maleimido)acetyl)piperazin-1-yl)methyl)-amatoxin;7′C-((4-(3-(maleimido)propanoyl)piperazin-1-yl)methyl)-amatoxin;7′C-((4-(4-(maleimido)butanoyl)piperazin-1-yl)methyl)-amatoxin;7′C-((4-(2-(2-(maleimido)acetamido)ethyl)piperidin-1-yl)methyl)-amatoxin;7′C-((4-(2-(4-(maleimido)butanamido)ethyl)piperidin-1-yl)methyl)-amatoxin;7′C-((4-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)ethyl)piperidin-1-yl)methyl)-amatoxin;7′C-((3-((6-(maleimido)hexanamido)methyl)azetidin-1-yl)methyl)-amatoxin;7′C-((3-(2-(6-(maleimido)hexanamido)ethyl)azetidin-1-yl)methyl)-amatoxin;7′C-((3-((4-((maleimido)methyl)cyclohexanecarboxamido)methyl)azetidin-1-yl)methyl)-amatoxin;7′C-((3-(2-(4-((maleimido)methyl)cyclohexanecarboxamido)ethyl)azetidin-1yl)methyl)-amatoxin;7′C-((3-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)ethyl)azetidin-1-yl)methyl)-amatoxin;7′C-(((2-(6-(maleimido)-N-methylhexanamido)ethyl)(methyl)amino)methyl)-amatoxin;7′C-(((4-(6-(maleimido)-N-methylhexanamido)butyl(methyl)amino)methyl)-amatoxin;7′C-((2-(2-(6-(maleimido)hexanamido)ethyl)aziridin-1-yl)methyl)-amatoxin;7′C-((2-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)ethyl)aziridin-1-yl)methyl)-amatoxin;7′C-((4-(6-(6-(2-(aminooxy)acetamido)hexanamido)hexanoyl)piperazin-1-yl)methyl)-amatoxin;7′C-((4-(1-(aminooxy)-2-oxo-6,9,12,15-tetraoxa-3-azaheptadecan-17-oyl)piperazin-1-yl)methyl)-amatoxin;7′C-((4-(2-(2-(aminooxy)acetamido)acetyl)piperazin-1-yl)methyl)-amatoxin;7′C-((4-(3-(2-(aminooxy)acetamido)propanoyl)piperazin-1-yl)methyl)-amatoxin;7′C-((4-(4-(2-(aminooxy)acetamido)butanoyl)piperazin-1-yl)methyl)-amatoxin;7′C-((4-(2-(6-(2-(aminooxy)acetamido)hexanamido)ethyl)piperidin-1-yl)methyl)-amatoxin;7′C-((4-(2-(2-(2-(aminooxy)acetamido)acetamido)ethyl)piperidin-1-yl)methyl)-amatoxin;7′C-((4-(2-(4-(2-(aminooxy)acetamido)butanamido)ethyl)piperidin-1-yl)methyl)-amatoxin;7′C-((4-(20-(aminooxy)-4,19-dioxo-6,9,12,15-tetraoxa-3,18-diazaicosyl)piperidin-1-yl)methyl)-amatoxin;7′C-(((2-(6-(2-(aminooxy)acetamido)-N-methylhexanamido)ethyl)(methyl)amino)methyl)-amatoxin;7′C-(((4-(6-(2-(aminooxy)acetamido)-N-methylhexanamido)butyl)(methyl)amino)methyl)-amatoxin;7′C-((3-((6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)methyl)pyrrolidin-1-yl)-S-methyl)-amatoxin;7′C-((3-((6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)-R-methyl)pyrrolidin-1-yl)methyl)-amatoxin;7′C-((4-(2-(2-bromoacetamido)ethyl)piperazin-1-yl)methyl)-amatoxin;7′C-((4-(2-(2-bromoacetamido)ethyl)piperidin-1-yl)methyl)-amatoxin;7′C-((4-(2-(3-(pyridine-2-yldisulfanyl)propanamido)ethyl)piperidin-1-yl)methyl)-amatoxin;6′O-(6-(6-(maleimido)hexanamido)hexyl)-amatoxin;6′O-(5-(4-((maleimido)methyl)cyclohexanecarboxamido)pentyl)-amatoxin;6′O-(2-((6-(maleimido)hexyl)oxy)-2-oxoethyl)-amatoxin;6′O-((6-(maleimido)hexyl)carbamoyl)-amatoxin;6′O-((6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexyl)carbamoyl)-amatoxin;6′O-(6-(2-bromoacetamido)hexyl)-amatoxin;7′C-(4-(6-(azido)hexanamido)piperidin-1-yl)-amatoxin;7′C-(4-(hex-5-ynoylamino)piperidin-1-yl)-amatoxin;7′C-(4-(2-(6-(maleimido)hexanamido)ethyl)piperazin-1-yl)-amatoxin;7′C-(4-(2-(6-(6-(maleimido)hexanamido)hexanamido)ethyl)piperazin-1-yl)-amatoxin;6′O-(6-(6-(11,12-didehydro-5,6-dihydro-dibenz[b,f]azocin-5-yl)-6-oxohexanamido)hexyl)-amatoxin;6′O-(6-(hex-5-ynoylamino)hexyl)-amatoxin;6′O-(6-(2-(aminooxy)acetylamido)hexyl)-amatoxin;6′O-((6-aminooxy)hexyl)-amatoxin; and6′O-(6-(2-iodoacetamido)hexyl)-amatoxin. The foregoing linkers, amongothers useful in conjunction with the compositions and methods describedherein, are described, for example, in US Patent Application PublicationNo. 2015/0218220, the disclosure of which is incorporated herein byreference in its entirety.

Additional cytotoxins that can be conjugated to antibodies,antigen-binding fragments thereof, and ligands that recognize and bindCD45 (such as CD45RO), CD135, CD34, CD90, and/or CD110 for use indirectly treating a cancer, autoimmune condition, or for conditioning apatient (e.g., a human patient) in preparation for hematopoietic stemcell transplant therapy include, without limitation, 5-ethynyluracil,abiraterone, acylfulvene, adecypenol, adozelesin, aldesleukin,altretamine, ambamustine, amidox, amifostine, aminolevulinic acid,amrubicin, amsacrine, anagrelide, anastrozole, andrographolide,angiogenesis inhibitors, antarelix, anti-dorsalizing morphogeneticprotein-1, antiandrogen, prostatic carcinoma, antiestrogen,antineoplaston, antisense oligonucleotides, aphidicolin glycinate,apoptosis gene modulators, apoptosis regulators, apurinic acid,asulacrine, atamestane, atrimustine, axinastatin 1, axinastatin 2,axinastatin 3, azasetron, azatoxin, azatyrosine, baccatin IIIderivatives, balanol, batimastat, BCR/ABL antagonists, benzochlorins,benzoylstaurosporine, beta lactam derivatives, beta-alethine,betaclamycin B, betulinic acid, bFGF inhibitors, bicalutamide,bisantrene, bisaziridinylspermine, bisnafide, bistratene A, bizelesin,breflate, bleomycin A2, bleomycin B2, bropirimine, budotitane,buthionine sulfoximine, calcipotriol, calphostin C, camptothecinderivatives (e.g., 10-hydroxy-camptothecin), capecitabine,carboxamide-amino-triazole, carboxyamidotriazole, carzelesin, caseinkinase inhibitors, castanospermine, cecropin B, cetrorelix, chlorins,chloroquinoxaline sulfonamide, cicaprost, cis-porphyrin, cladribine,clomifene and analogues thereof, clotrimazole, collismycin A,collismycin B, combretastatin A4, combretastatin analogues, conagenin,crambescidin 816, crisnatol, cryptophycin 8, cryptophycin A derivatives,curacin A, cyclopentanthraquinones, cycloplatam, cypemycin, cytarabineocfosfate, cytolytic factor, cytostatin, dacliximab, decitabine,dehydrodidemnin B, 2′deoxycoformycin (DCF), deslorelin, dexifosfamide,dexrazoxane, dexverapamil, diaziquone, didemnin B, didox,diethylnorspermine, dihydro-5-azacytidine, dihydrotaxol, dioxamycin,diphenyl spiromustine, discodermolide, docosanol, dolasetron,doxifluridine, droloxifene, dronabinol, duocarmycin SA, ebselen,ecomustine, edelfosine, edrecolomab, eflornithine, elemene, emitefur,epothilones, epithilones, epristeride, estramustine and analoguesthereof, etoposide, etoposide 4′-phosphate (also referred to asetopofos), exemestane, fadrozole, fazarabine, fenretinide, filgrastim,finasteride, flavopiridol, flezelastine, fluasterone, fludarabine,fluorodaunorunicin hydrochloride, forfenimex, formestane, fostriecin,fotemustine, gadolinium texaphyrin, gallium nitrate, galocitabine,ganirelix, gelatinase inhibitors, gemcitabine, glutathione inhibitors,hepsulfam, homoharringtonine (HHT), hypericin, ibandronic acid,idoxifene, idramantone, ilmofosine, ilomastat, imidazoacridones,imiquimod, immunostimulant peptides, iobenguane, iododoxorubicin,ipomeanol, irinotecan, iroplact, irsogladine, isobengazole,jasplakinolide, kahalalide F, lamellarin-N triacetate, lanreotide,leinamycin, lenograstim, lentinan sulfate, leptolstatin, letrozole,lipophilic platinum compounds, lissoclinamide 7, lobaplatin, lometrexol,lonidamine, losoxantrone, loxoribine, lurtotecan, lutetium texaphyrin,lysofylline, masoprocol, maspin, matrix metalloproteinase inhibitors,menogaril, rnerbarone, meterelin, methioninase, metoclopramide, MIFinhibitor, ifepristone, miltefosine, mirimostim, mithracin, mitoguazone,mitolactol, mitomycin and analogues thereof, mitonafide, mitoxantrone,mofarotene, molgramostim, mycaperoxide B, myriaporone, N-acetyldinaline,N-substituted benzamides, nafarelin, nagrestip, napavin, naphterpin,nartograstim, nedaplatin, nemorubicin, neridronic acid, nilutamide,nisamycin, nitrullyn, octreotide, okicenone, onapristone, ondansetron,oracin, ormaplatin, oxaliplatin, oxaunomycin, paclitaxel and analoguesthereof, palauamine, palmitoylrhizoxin, pamidronic acid, panaxytriol,panomifene, parabactin, pazelliptine, pegaspargase, peldesine, pentosanpolysulfate sodium, pentostatin, pentrozole, perflubron, perfosfamide,phenazinomycin, picibanil, pirarubicin, piritrexim, podophyllotoxin,porfiromycin, purine nucleoside phosphorylase inhibitors, raltitrexed,rhizoxin, rogletimide, rohitukine, rubiginone B1, ruboxyl, safingol,saintopin, sarcophytol A, sargramostim, sobuzoxane, sonermin, sparfosicacid, spicamycin D, spiromustine, stipiamide, sulfinosine, tallimustine,tegafur, temozolomide, teniposide, thaliblastine, thiocoraline,tirapazamine, topotecan, topsentin, triciribine, trimetrexate, veramine,vinorelbine, vinxaltine, vorozole, zeniplatin, and zilascorb, amongothers.

Linkers for Chemical Conjugation

A variety of linkers can be used to conjugate antibodies,antigen-binding fragments, and ligands described herein (e.g.,antibodies, antigen-binding fragments thereof, and ligands thatrecognize and bind CD45 (such as CD45RO), CD135, CD34, CD90, and/orCD110) with a cytotoxic molecule. Linkers include those that may becleaved, for instance, by enzymatic hydrolysis, photolysis, hydrolysisunder acidic conditions, hydrolysis under basic conditions, oxidation,disulfide reduction, nucleophilic cleavage, or organometallic cleavage(see, for example, Leriche et al., Bioorg. Med. Chem., 20:571-582, 2012,the disclosure of which is incorporated herein by reference as itpertains to linkers suitable for covalent conjugation). Examples oflinkers useful for the synthesis of drug-antibody conjugates anddrug-ligand conjugates include those that contain electrophiles, such asMichael acceptors (e.g., maleimides), activated esters,electron-deficient carbonyl compounds, and aldehydes, among others,suitable for reaction with nucleophilic substituents present withinantibodies or antigen-binding fragments, such as amine and thiolmoieties. For instance, linkers suitable for the synthesis ofdrug-antibody conjugates and drug-ligand conjugates include, withoutlimitation, succinimidyl 4-(N-maleimidomethyl)-cyclohexane-L-carboxylate(SMCC), N-succinimidyl iodoacetate (SIA), sulfo-SMCC,m-maleimidobenzoyl-N-hydroxysuccinimidyl ester (MBS), sulfo-MBS, andsuccinimidyl iodoacetate, among others described, for instance, Liu etal., 18:690-697, 1979, the disclosure of which is incorporated herein byreference as it pertains to linkers for chemical conjugation. Additionallinkers include the non-cleavable maleimidocaproyl linkers, which areparticularly useful for the conjugation of microtubule-disrupting agentssuch as auristatins, are described by Doronina et al., BioconjugateChem. 17:14-24, 2006, the disclosure of which is incorporated herein byreference as it pertains to linkers for chemical conjugation. Additionallinkers suitable for the synthesis of drug-antibody conjugates anddrug-ligand conjugates as described herein include those capable ofreleasing a cytotoxin by a 1,6-elimination process, such asp-aminobenzyl alcohol (PABC), 6-maleimidohexanoic acid, pH-sensitivecarbonates, and other reagents described in Jain et al., Pharm. Res.32:3526-3540, 2015, the disclosure of which is incorporated herein byreference in its entirety.

Linkers that can be used to conjugate an antibody, antigen-bindingfragment thereof, or ligand to a cytotoxic agent include those that arecovalently bound to the cytotoxic agent on one end of the linker and, onthe other end of the linker, contain a chemical moiety formed from acoupling reaction between a reactive substituent present on the linkerand a reactive substituent present within the antibody, antigen-bindingfragment thereof, or ligand that binds CD45 (such as CD45RO), CD135,CD34, CD90, and/or CD110. Reactive substituents that may be presentwithin an antibody, antigen-binding fragment thereof, or ligand thatbinds CD45 (such as CD45RO), CD135, CD34, CD90, and/or CD110 include,without limitation, hydroxyl moieties of serine, threonine, and tyrosineresidues; amino moieties of lysine residues; carboxyl moieties ofaspartic acid and glutamic acid residues; and thiol moieties of cysteineresidues, as well as propargyl, azido, haloaryl (e.g., fluoroaryl),haloheteroaryl (e.g., fluoroheteroaryl), haloalkyl, and haloheteroalkylmoieties of non-naturally occurring amino acids. Linkers useful inconjunction with the antibody-drug and ligand-drug conjugates describedherein include, without limitation, linkers containing chemical moietiesformed by coupling reactions as depicted in Table 2, below. Curved linesdesignate points of attachment to the antibody, antigen-bindingfragment, or ligand and the cytotoxic molecule, respectively.

TABLE 2 Exemplary chemical moieties formed by coupling reactions in theformation of antibody-drug and ligand-drug conjugates Exemplary CouplingReactions Chemical Moiety Formed by Coupling Reactions [3 + 2]Cycloaddition

[3 + 2] Cycloaddition

[3 + 2] Cycloaddition, Esterification

[3 + 2] Cycloaddition, Esterification

[3 + 2] Cycloaddition, Esterification

[3 + 2] Cycloaddition, Esterification

[3 + 2] Cycloaddition, Esterification

[3 + 2] Cycloaddition, Esterification

[3 + 2] Cycloaddition, Esterification

[3 + 2] Cycloaddition, Esterification

[3 + 2] Cycloaddition, Esterification

[3 + 2] Cycloaddition, Esterification

[3 + 2] Cycloaddition, Esterification

[3 + 2] Cycloaddition, Etherification

[3 + 2] Cycloaddition

Michael addition

Michael addition

Imine condensation, Amidation

Imine condensation

Disulfide formation

Thiol alkylation

Condensation, Michael addition

Methods of Treatment

As described herein, hematopoietic stem cell transplant therapy can beadministered to a subject in need of treatment so as to populate orre-populate one or more blood cell types. Hematopoietic stem cellsgenerally exhibit multi-potency, and can thus differentiate intomultiple different blood lineages including, but not limited to,granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils),erythrocytes (e.g., reticulocytes, erythrocytes), thrombocytes (e.g.,megakaryoblasts, platelet producing megakaryocytes, platelets),monocytes (e.g., monocytes, macrophages), dendritic cells, microglia,osteoclasts, and lymphocytes (e.g., NK cells, B-cells and T-cells).Hematopoietic stem cells are additionally capable of self-renewal, andcan thus give rise to daughter cells that have equivalent potential asthe mother cell, and also feature the capacity to be reintroduced into atransplant recipient whereupon they home to the hematopoietic stem cellniche and re-establish productive and sustained hematopoiesis.

Hematopoietic stem cells can thus be administered to a patient defectiveor deficient in one or more cell types of the hematopoietic lineage inorder to re-constitute the defective or deficient population of cells invivo, thereby treating the pathology associated with the defect ordepletion in the endogenous blood cell population. The compositions andmethods described herein can thus be used to treat a non-malignanthemoglobinopathy (e.g., a hemoglobinopathy selected from the groupconsisting of sickle cell anemia, thalassemia, Fanconi anemia, aplasticanemia, and Wiskott-Aldrich syndrome). Additionally or alternatively,the compositions and methods described herein can be used to treat animmunodeficiency, such as a congenital immunodeficiency. Additionally oralternatively, the compositions and methods described herein can be usedto treat an acquired immunodeficiency (e.g., an acquiredimmunodeficiency selected from the group consisting of HIV and AIDS).The compositions and methods described herein can be used to treat ametabolic disorder (e.g., a metabolic disorder selected from the groupconsisting of glycogen storage diseases, mucopolysaccharidoses,Gaucher's Disease, Hurlers Disease, sphingolipidoses, and metachromaticleukodystrophy).

Additionally or alternatively, the compositions and methods describedherein can be used to treat a malignancy or proliferative disorder, suchas a hematologic cancer, myeloproliferative disease. In the case ofcancer treatment, the compositions and methods described herein may beadministered to a patient so as to deplete a population of endogenoushematopoietic stem cells prior to hematopoietic stem celltransplantation therapy, in which case the transplanted cells can hometo a niche created by the endogenous cell depletion step and establishproductive hematopoiesis. This, in turn, can re-constitute a populationof cells depleted during cancer cell eradication, such as duringsystemic chemotherapy. Exemplary hematological cancers that can betreated using the compositions and methods described heein include,without limitation, acute myeloid leukemia, acute lymphoid leukemia,chronic myeloid leukemia, chronic lymphoid leukemia, multiple myeloma,diffuse large B-cell lymphoma, and non-Hodgkin's lymphoma, as well asother cancerous conditions, including neuroblastoma.

Additional diseases that can be treated with the compositions andmethods described herein include, without limitation, adenosinedeaminase deficiency and severe combined immunodeficiency, hyperimmunoglobulin M syndrome, Chediak-Higashi disease, hereditarylymphohistiocytosis, osteopetrosis, osteogenesis imperfecta, storagediseases, thalassemia major, systemic sclerosis, systemic lupuserythematosus, multiple sclerosis, and juvenile rheumatoid arthritis.

The antibodies, antigen-binding fragments thereof, ligands, andconjugates described herein may be used to induce solid organ transplanttolerance. For instance, the compositions and methods described hereinmay be used to deplete or ablate a population of cells from a targettissue (e.g., to deplete hematopoietic stem cells from the bone marrowstem cell niche). Following such depletion of cells from the targettissues, a population of stem or progenitor cells from an organ donor(e.g., hematopoietic stem cells from the organ donor) may beadministered to the transplant recipient, and following the engraftmentof such stem or progenitor cells, a temporary or stable mixed chimerismmay be achieved, thereby enabling long-term transplant organ tolerancewithout the need for further immunosuppressive agents. For example, thecompositions and methods described herein may be used to inducetransplant tolerance in a solid organ transplant recipient (e.g., akidney transplant, lung transplant, liver transplant, and hearttransplant, among others). The compositions and methods described hereinare well-suited for use in connection the induction of solid organtransplant tolerance, for instance, because a low percentage temporaryor stable donor engraftment is sufficient to induce long-term toleranceof the transplanted organ.

In addition, the compositions and methods described herein can be usedto treat cancers directly, such as cancers characterized by cells thatare CD45+, CD135+, CD34+, CD90+, or CD110+. For instance, thecompositions and methods described herein can be used to treat leukemia,particularly in patients that exhibit CD45+, CD135+, CD34+, CD90+, orCD110+ leukemic cells. By depleting CD45+, CD135+, CD34+, CD90+, orCD110+ cancerous cells, such as leukemic cells, the compositions andmethods described herein can be used to treat various cancers directly.Exemplary cancers that may be treated in this fashion includehematological cancers, such as acute myeloid leukemia, acute lymphoidleukemia, chronic myeloid leukemia, chronic lymphoid leukemia, multiplemyeloma, diffuse large B-cell lymphoma, and non-Hodgkin's lymphoma,

In addition, the compositions and methods described herein can be usedto treat autoimmune disorders. For instance, an antibody,antigen-binding fragment thereof, or ligand can be administered to asubject, such as a human patient suffering from an autoimmune disorder,so as to kill a CD45+, CD135+, CD34+, CD90+, or CD110+ immune cell. TheCD45+, CD135+, CD34+, CD90+, or CD110+ immune cell may be anautoreactive lymphocyte, such as a T-cell that expresses a T-cellreceptor that specifically binds, and mounts an immune response against,a self antigen. By depleting self-reactive, CD45+, CD135+, CD34+, CD90+,or CD110+ cells, the compositions and methods described herein can beused to treat autoimmune pathologies, such as those described below.Additionally or alternatively, the compositions and methods describedherein can be used to treat an autoimmune disease by depleting apopulation of endogenous hematopoietic stem cells prior to hematopoieticstem cell transplantation therapy, in which case the transplanted cellscan home to a niche created by the endogenous cell depletion step andestablish productive hematopoiesis. This, in turn, can re-constitute apopulation of cells depleted during autoimmune cell eradication.

Autoimmune diseases that can be treated using the compositions andmethods described herein include, without limitation, psoriasis,psoriatic arthritis, Type 1 diabetes mellitus (Type 1 diabetes),rheumatoid arthritis (RA), human systemic lupus (SLE), multiplesclerosis (MS), inflammatory bowel disease (IBD), lymphocytic colitis,acute disseminated encephalomyelitis (ADEM), Addison's disease, alopeciauniversalis, ankylosing spondylitisis, antiphospholipid antibodysyndrome (APS), aplastic anemia, autoimmune hemolytic anemia, autoimmunehepatitis, autoimmune inner ear disease (AIED), autoimmunelymphoproliferative syndrome (ALPS), autoimmune oophoritis, Balodisease, Behcet's disease, bullous pemphigoid, cardiomyopathy, Chagas'disease, chronic fatigue immune dysfunction syndrome (CFIDS), chronicinflammatory demyelinating polyneuropathy, Crohn's disease, cicatricalpemphigoid, coeliac sprue-dermatitis herpetiformis, cold agglutinindisease, CREST syndrome, Degos disease, discoid lupus, dysautonomia,endometriosis, essential mixed cryoglobulinemia,fibromyalgia-fibromyositis, Goodpasture's syndrome, Grave's disease,Guillain-Barre syndrome (GBS), Hashimoto's thyroiditis, Hidradenitissuppurativa, idiopathic and/or acute thrombocytopenic purpura,idiopathic pulmonary fibrosis, IgA neuropathy, interstitial cystitis,juvenile arthritis, Kawasaki's disease, lichen planus, Lyme disease,Meniere disease, mixed connective tissue disease (MCTD), myastheniagravis, neuromyotonia, opsoclonus myoclonus syndrome (OMS), opticneuritis, Ord's thyroiditis, pemphigus vulgaris, pernicious anemia,polychondritis, polymyositis and dermatomyositis, primary biliarycirrhosis, polyarteritis nodosa, polyglandular syndromes, polymyalgiarheumatica, primary agammaglobulinemia, Raynaud phenomenon, Reiter'ssyndrome, rheumatic fever, sarcoidosis, scleroderma, Sjögren's syndrome,stiff person syndrome, Takayasu's arteritis, temporal arteritis (alsoknown as “giant cell arteritis”), ulcerative colitis, collagenouscolitis, uveitis, vasculitis, vitiligo, vulvodynia (“vulvarvestibulitis”), and Wegener's granulomatosis.

Routes of Administration and Dosing

Antibodies, antigen-binding fragments thereof, and ligands describedherein can be administered to a patient (e.g., a human patient sufferingfrom cancer, an autoimmune disease, or in need of hematopoietic stemcell transplant therapy) in a variety of dosage forms. For instance,antibodies, antigen-binding fragments thereof, and ligands describedherein can be administered to a patient suffering from cancer, anautoimmune disease, or in need of hematopoietic stem cell transplanttherapy in the form of an aqueous solution, such as an aqueous solutioncontaining one or more pharmaceutically acceptable excipients.Pharmaceutically acceptable excipients for use with the compositions andmethods described herein include viscosity-modifying agents. The aqueoussolution may be sterilized using techniques known in the art.

The antibodies, antigen-binding fragments, and ligands described hereinmay be administered by a variety of routes, such as orally,transdermally, subcutaneously, intranasally, intravenously,intramuscularly, intraocularly, or parenterally. The most suitable routefor administration in any given case will depend on the particularantibody, antigen-binding fragment, or ligand administered, the patient,pharmaceutical formulation methods, administration methods (e.g.,administration time and administration route), the patient's age, bodyweight, sex, severity of the diseases being treated, the patient's diet,and the patient's excretion rate.

The effective dose of an antibody, antigen-binding fragment thereof, orligand described herein can range, for example from about 0.001 to about100 mg/kg of body weight per single (e.g., bolus) administration,multiple administrations, or continuous administration, or to achieve anoptimal serum concentration (e.g., a serum concentration of 0.0001-5000μg/mL) of the antibody, antigen-binding fragment thereof, or ligand. Thedose may be administered one or more times (e.g., 2-10 times) per day,week, or month to a subject (e.g., a human) suffering from cancer, anautoimmune disease, or undergoing conditioning therapy in preparationfor receipt of a hematopoietic stem cell transplant. In the case of aconditioning procedure prior to hematopoietic stem cell transplantation,the antibody, antigen-binding fragment thereof, or ligand can beadministered to the patient at a time that optimally promotesengraftment of the exogenous hematopoietic stem cells, for instance,from 1 hour to 1 week (e.g., 1 hour, 2 hours, 3 hours, 4 hours, 5 hours,6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20hours, 21 hours, 22 hours, 23 hours, 24 hours, 2 days, 3 days, 4 days, 5days, 6 days, or 7 days) or more prior to administration of theexogenous hematopoietic stem cell transplant.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a description of how the compositions and methodsdescribed herein may be used, made, and evaluated, and are intended tobe purely exemplary of the invention and are not intended to limit thescope of what the inventors regard as their invention.

Example 1. Administration of an Anti-CD45 Antibody to a Human Patient inPreparation for Hematopoietic Stem Cell Transplant Therapy

According to the methods disclosed herein, a physician of skill in theart can condition a patient, such as a human patient, so as to promotethe engraftment of exogenous hematopoietic stem cell grafts prior tohematopoietic stem cell transplant therapy. To this end, a physician ofskill in the art can administer to the human patient an antibody orantigen-binding fragment thereof capable of binding an antigen expressedby hematopoietic stem cells, such as an antibody or antigen-bidingfragment thereof that binds CD45 (for example, an antibody orantigen-binding fragment thereof that binds CD45RO). The antibody may becovalently conjugated to a toxin, such as a cytotoxic molecule describedherein or known in the art. For instance, an anti-CD45 antibody orantigen-binding fragment thereof (such as an anti-CD45RO antibody orantigen-binding fragment thereof) can be covalently conjugated to acytotoxin, such as pseudomonas exotoxin A, deBouganin, diphtheria toxin,an amatoxin, such as α-amanitin, saporin, maytansine, a maytansinoid, anauristatin, an anthracycline, a calicheamicin, irinotecan, SN-38, aduocarmycin, a pyrrolobenzodiazepine, a pyrrolobenzodiazepine dimer, anindolinobenzodiazepine, an indolinobenzodiazepine dimer, or a variantthereof. This conjugation can be performed using covalent bond-formingtechniques described herein or known in the art. The antibody,antigen-binding fragment thereof, or drug-antibody conjugate cansubsequently be administered to the patient, for example, by intravenousadministration, prior to transplantation of exogenous hematopoietic stemcells (such as autologous, syngeneic, or allogeneic hematopoietic stemcells) to the patient.

The anti-CD45 (e.g., anti-CD45RO) antibody, antigen-binding fragmentthereof, or drug-antibody conjugate can be administered in an amountsufficient to reduce the quantity of endogenous hematopoietic stemcells, for example, by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%,or more prior to hematopoietic stem cell transplant therapy. Thereduction in hematopoietic stem cell count can be monitored usingconventional techniques known in the art, such as by FACS analysis ofcells expressing characteristic hematopoietic stem cell surface antigensin a blood sample withdrawn from the patient at varying intervals duringconditioning therapy. For instance, a physician of skill in the art canwithdraw a blood sample from the patient at various time points duringconditioning therapy and determine the extent of endogenoushematopoietic stem cell reduction by conducting a FACS analysis toelucidate the relative concentrations of hematopoietic stem cells in thesample using antibodies that bind to hematopoietic stem cell markerantigens. According to some embodiments, when the concentration ofhematopoietic stem cells has reached a minimum value in response toconditioning therapy with an anti-CD45 (e.g., anti-CD45RO) antibody,antigen-binding fragment thereof, or drug-antibody conjugate, thephysician may conclude the conditioning therapy, and may begin preparingthe patient for hematopoietic stem cell transplant therapy.

The anti-CD45 (e.g., anti-CD45RO) antibody, antigen-binding fragmentthereof, or drug-antibody conjugate can be administered to the patientin an aqueous solution containing one or more pharmaceuticallyacceptable excipients, such as a viscosity-modifying agent. The aqueoussolution may be sterilized using techniques described herein or known inthe art. The antibody, antigen-binding fragment thereof, ordrug-antibody conjugate can be administered to the patient at a dosageof, for example, from 0.001 mg/kg to 100 mg/kg prior to administrationof a hematopoietic stem cell graft to the patient. The antibody,antigen-binding fragment thereof, or drug-antibody conjugate can beadministered to the patient at a time that optimally promotesengraftment of the exogenous hematopoietic stem cells, for instance,from 1 hour to 1 week (e.g., 1 hour, 2 hours, 3 hours, 4 hours, 5 hours,6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20hours, 21 hours, 22 hours, 23 hours, 24 hours, 2 days, 3 days, 4 days, 5days, 6 days, or 7 days) or more prior to administration of theexogenous hematopoietic stem cell transplant.

Following the conclusion of conditioning therapy, the patient may thenreceive an infusion (e.g., an intravenous infusion) of exogenoushematopoietic stem cells, such as from the same physician that performedthe conditioning therapy or from a different physician. The physicianmay administer the patient an infusion of autologous, syngeneic, orallogeneic hematopoietic stem cells, for instance, at a dosage of from1×10³ to 1×10⁹ hematopoietic stem cells/kg. The physician may monitorthe engraftment of the hematopoietic stem cell transplant, for example,by withdrawing a blood sample from the patient and determining theincrease in concentration of hematopoietic stem cells or cells of thehematopoietic lineage (such as megakaryocytes, thrombocytes, platelets,erythrocytes, mast cells, myeoblasts, basophils, neutrophils,eosinophils, microglia, granulocytes, monocytes, osteoclasts,antigen-presenting cells, macrophages, dendritic cells, natural killercells, T-lymphocytes, and B-lymphocytes) following administration of thetransplant. This analysis may be conducted, for example, from 1 hour to6 months, or more, following hematopoietic stem cell transplant therapy(e.g., 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22hours, 23 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23weeks, 24 weeks, or more). A finding that the concentration ofhematopoietic stem cells or cells of the hematopoietic lineage hasincreased (e.g., by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, or more) following thetransplant therapy relative to the concentration of the correspondingcell type prior to transplant therapy provides one indication thattreatment with the anti-CD45 (e.g., anti-CD45RO) antibody,antigen-binding fragment thereof, or drug-antibody conjugate hassuccessfully promoted engraftment of the transplanted hematopoietic stemcell graft.

Example 2. Administration of an Anti-CD135 Antibody to a Human Patientin Preparation for Hematopoietic Stem Cell Transplant Therapy

Using the methods disclosed herein, a physician of skill in the art canadminister to a human patient in need of hematopoietic stem celltransplant therapy an antibody or antigen-binding fragment thereofcapable of binding an antigen expressed by hematopoietic stem cells,such as an antibody or antigen-biding fragment thereof that binds CD135.In this fashion, a population of endogenous hematopoietic stem cells canbe depleted prior to administration of an exogenous hematopoietic stemcell graft so as to promote engraftment of the hematopoietic stem cellgraft. The antibody may be covalently conjugated to a toxin, such as acytotoxic molecule described herein or known in the art. For instance,an anti-CD135 antibody or antigen-binding fragment thereof can becovalently conjugated to a cytotoxin, such as pseudomonas exotoxin A,deBouganin, diphtheria toxin, an amatoxin, such as α-amanitin, saporin,maytansine, a maytansinoid, an auristatin, an anthracycline, acalicheamicin, irinotecan, SN-38, a duocarmycin, apyrrolobenzodiazepine, a pyrrolobenzodiazepine dimer, anindolinobenzodiazepine, an indolinobenzodiazepine dimer, or a variantthereof. This conjugation can be performed using covalent bond-formingtechniques described herein or known in the art. The antibody,antigen-binding fragment thereof, or drug-antibody conjugate cansubsequently be administered to the patient, for example, by intravenousadministration, prior to transplantation of exogenous hematopoietic stemcells (such as autologous, syngeneic, or allogeneic hematopoietic stemcells) to the patient.

The anti-CD135 antibody, antigen-binding fragment thereof, ordrug-antibody conjugate can be administered in an amount sufficient toreduce the quantity of endogenous hematopoietic stem cells, for example,by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more prior tohematopoietic stem cell transplant therapy. The reduction inhematopoietic stem cell count can be monitored using conventionaltechniques known in the art, such as by FACS analysis of cellsexpressing characteristic hematopoietic stem cell surface antigens in ablood sample withdrawn from the patient at varying intervals duringconditioning therapy. For instance, a physician of skill in the art canwithdraw a blood sample from the patient at various time points duringconditioning therapy and determine the extent of endogenoushematopoietic stem cell reduction by conducting a FACS analysis toelucidate the relative concentrations of hematopoietic stem cells in thesample using antibodies that bind to hematopoietic stem cell markerantigens. According to some embodiments, when the concentration ofhematopoietic stem cells has reached a minimum value in response toconditioning therapy with an anti-CD135 antibody, antigen-bindingfragment thereof, or drug-antibody conjugate, the physician may concludethe conditioning therapy, and may begin preparing the patient forhematopoietic stem cell transplant therapy.

The anti-CD135 antibody, antigen-binding fragment thereof, ordrug-antibody conjugate can be administered to the patient in an aqueoussolution containing one or more pharmaceutically acceptable excipients,such as a viscosity-modifying agent. The aqueous solution may besterilized using techniques described herein or known in the art. Theantibody, antigen-binding fragment thereof, or drug-antibody conjugatecan be administered to the patient at a dosage of, for example, from0.001 mg/kg to 100 mg/kg prior to administration of a hematopoietic stemcell graft to the patient. The antibody, antigen-binding fragmentthereof, or drug-antibody conjugate can be administered to the patientat a time that optimally promotes engraftment of the exogenoushematopoietic stem cells, for instance, from 1 hour to 1 week (e.g., 1hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days) ormore prior to administration of the exogenous hematopoietic stem celltransplant.

Following the conclusion of conditioning therapy, the patient may thenreceive an infusion (e.g., an intravenous infusion) of exogenoushematopoietic stem cells, such as from the same physician that performedthe conditioning therapy or from a different physician. The physicianmay administer the patient an infusion of autologous, syngeneic, orallogeneic hematopoietic stem cells, for instance, at a dosage of from1×10³ to 1×10⁹ hematopoietic stem cells/kg. The physician may monitorthe engraftment of the hematopoietic stem cell transplant, for example,by withdrawing a blood sample from the patient and determining theincrease in concentration of hematopoietic stem cells or cells of thehematopoietic lineage (such as megakaryocytes, thrombocytes, platelets,erythrocytes, mast cells, myeoblasts, basophils, neutrophils,eosinophils, microglia, granulocytes, monocytes, osteoclasts,antigen-presenting cells, macrophages, dendritic cells, natural killercells, T-lymphocytes, and B-lymphocytes) following administration of thetransplant. This analysis may be conducted, for example, from 1 hour to6 months, or more, following hematopoietic stem cell transplant therapy(e.g., 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22hours, 23 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23weeks, 24 weeks, or more). A finding that the concentration ofhematopoietic stem cells or cells of the hematopoietic lineage hasincreased (e.g., by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, or more) following thetransplant therapy relative to the concentration of the correspondingcell type prior to transplant therapy provides one indication thattreatment with the anti-CD135 antibody, antigen-binding fragmentthereof, or drug-antibody conjugate has successfully promotedengraftment of the transplanted hematopoietic stem cell graft.

Example 3. Administration of an Anti-CD34 Antibody to a Human Patient inPreparation for Hematopoietic Stem Cell Transplant Therapy

Using the methods disclosed herein, a physician of skill in the art canadminister to a human patient in need of hematopoietic stem celltransplant therapy an antibody or antigen-binding fragment thereofcapable of binding an antigen expressed by hematopoietic stem cells,such as an antibody or antigen-biding fragment thereof that binds CD34.In this way, a population of endogenous hematopoietic stem cells can bedepleted prior to administration of an exogenous hematopoietic stem cellgraft so as to promote engraftment of the hematopoietic stem cell graft.The antibody may be covalently conjugated to a toxin, such as acytotoxic molecule described herein or known in the art. For instance,an anti-CD34 antibody or antigen-binding fragment thereof can becovalently conjugated to a cytotoxin, such as pseudomonas exotoxin A,deBouganin, diphtheria toxin, an amatoxin, such as α-amanitin, saporin,maytansine, a maytansinoid, an auristatin, an anthracycline, acalicheamicin, irinotecan, SN-38, a duocarmycin, apyrrolobenzodiazepine, a pyrrolobenzodiazepine dimer, anindolinobenzodiazepine, an indolinobenzodiazepine dimer, or a variantthereof. This conjugation can be performed using covalent bond-formingtechniques described herein or known in the art. The antibody,antigen-binding fragment thereof, or drug-antibody conjugate cansubsequently be administered to the patient, for example, by intravenousadministration, prior to transplantation of exogenous hematopoietic stemcells (such as autologous, syngeneic, or allogeneic hematopoietic stemcells) to the patient.

The anti-CD34 antibody, antigen-binding fragment thereof, ordrug-antibody conjugate can be administered in an amount sufficient toreduce the quantity of endogenous hematopoietic stem cells, for example,by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more prior tohematopoietic stem cell transplant therapy. The reduction inhematopoietic stem cell count can be monitored using conventionaltechniques known in the art, such as by FACS analysis of cellsexpressing characteristic hematopoietic stem cell surface antigens in ablood sample withdrawn from the patient at varying intervals duringconditioning therapy. For instance, a physician of skill in the art canwithdraw a blood sample from the patient at various time points duringconditioning therapy and determine the extent of endogenoushematopoietic stem cell reduction by conducting a FACS analysis toelucidate the relative concentrations of hematopoietic stem cells in thesample using antibodies that bind to hematopoietic stem cell markerantigens. According to some embodiments, when the concentration ofhematopoietic stem cells has reached a minimum value in response toconditioning therapy with an anti-CD34 antibody, antigen-bindingfragment thereof, or drug-antibody conjugate, the physician may concludethe conditioning therapy, and may begin preparing the patient forhematopoietic stem cell transplant therapy.

The anti-CD34 antibody, antigen-binding fragment thereof, ordrug-antibody conjugate can be administered to the patient in an aqueoussolution containing one or more pharmaceutically acceptable excipients,such as a viscosity-modifying agent. The aqueous solution may besterilized using techniques described herein or known in the art. Theantibody, antigen-binding fragment thereof, or drug-antibody conjugatecan be administered to the patient at a dosage of, for example, from0.001 mg/kg to 100 mg/kg prior to administration of a hematopoietic stemcell graft to the patient. The antibody, antigen-binding fragmentthereof, or drug-antibody conjugate can be administered to the patientat a time that optimally promotes engraftment of the exogenoushematopoietic stem cells, for instance, from 1 hour to 1 week (e.g., 1hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days) ormore prior to administration of the exogenous hematopoietic stem celltransplant.

Following the conclusion of conditioning therapy, the patient may thenreceive an infusion (e.g., an intravenous infusion) of exogenoushematopoietic stem cells, such as from the same physician that performedthe conditioning therapy or from a different physician. The physicianmay administer the patient an infusion of autologous, syngeneic, orallogeneic hematopoietic stem cells, for instance, at a dosage of from1×10³ to 1×10⁹ hematopoietic stem cells/kg. The physician may monitorthe engraftment of the hematopoietic stem cell transplant, for example,by withdrawing a blood sample from the patient and determining theincrease in concentration of hematopoietic stem cells or cells of thehematopoietic lineage (such as megakaryocytes, thrombocytes, platelets,erythrocytes, mast cells, myeoblasts, basophils, neutrophils,eosinophils, microglia, granulocytes, monocytes, osteoclasts,antigen-presenting cells, macrophages, dendritic cells, natural killercells, T-lymphocytes, and B-lymphocytes) following administration of thetransplant. This analysis may be conducted, for example, from 1 hour to6 months, or more, following hematopoietic stem cell transplant therapy(e.g., 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22hours, 23 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23weeks, 24 weeks, or more). A finding that the concentration ofhematopoietic stem cells or cells of the hematopoietic lineage hasincreased (e.g., by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, or more) following thetransplant therapy relative to the concentration of the correspondingcell type prior to transplant therapy provides one indication thattreatment with the anti-CD34 antibody, antigen-binding fragment thereof,or drug-antibody conjugate has successfully promoted engraftment of thetransplanted hematopoietic stem cell graft.

Example 4. Administration of an Anti-CD90 Antibody to a Human Patient inPreparation for Hematopoietic Stem Cell Transplant Therapy

Using the methods disclosed herein, a physician of skill in the art canadminister to a human patient in need of hematopoietic stem celltransplant therapy an antibody or antigen-binding fragment thereofcapable of binding an antigen expressed by hematopoietic stem cells,such as an antibody or antigen-biding fragment thereof that binds CD90.In this way, a population of endogenous hematopoietic stem cells can bedepleted prior to administration of an exogenous hematopoietic stem cellgraft so as to promote engraftment of the hematopoietic stem cell graft.The antibody may be covalently conjugated to a toxin, such as acytotoxic molecule described herein or known in the art. For instance,an anti-CD90 antibody or antigen-binding fragment thereof (such as ananti-GNNK+ CD90 antibody or antigen-binding fragment thereof) can becovalently conjugated to a cytotoxin, such as pseudomonas exotoxin A,deBouganin, diphtheria toxin, an amatoxin, such as α-amanitin, saporin,maytansine, a maytansinoid, an auristatin, an anthracycline, acalicheamicin, irinotecan, SN-38, a duocarmycin, apyrrolobenzodiazepine, a pyrrolobenzodiazepine dimer, anindolinobenzodiazepine, an indolinobenzodiazepine dimer, or a variantthereof. This conjugation can be performed using covalent bond-formingtechniques described herein or known in the art. The antibody,antigen-binding fragment thereof, or drug-antibody conjugate cansubsequently be administered to the patient, for example, by intravenousadministration, prior to transplantation of exogenous hematopoietic stemcells (such as autologous, syngeneic, or allogeneic hematopoietic stemcells) to the patient.

The anti-CD90 antibody, antigen-binding fragment thereof, ordrug-antibody conjugate can be administered in an amount sufficient toreduce the quantity of endogenous hematopoietic stem cells, for example,by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more prior tohematopoietic stem cell transplant therapy. The reduction inhematopoietic stem cell count can be monitored using conventionaltechniques known in the art, such as by FACS analysis of cellsexpressing characteristic hematopoietic stem cell surface antigens in ablood sample withdrawn from the patient at varying intervals duringconditioning therapy. For instance, a physician of skill in the art canwithdraw a blood sample from the patient at various time points duringconditioning therapy and determine the extent of endogenoushematopoietic stem cell reduction by conducting a FACS analysis toelucidate the relative concentrations of hematopoietic stem cells in thesample using antibodies that bind to hematopoietic stem cell markerantigens. According to some embodiments, when the concentration ofhematopoietic stem cells has reached a minimum value in response toconditioning therapy with an anti-CD90 antibody, antigen-bindingfragment thereof, or drug-antibody conjugate, the physician may concludethe conditioning therapy, and may begin preparing the patient forhematopoietic stem cell transplant therapy.

The anti-CD90 antibody, antigen-binding fragment thereof, ordrug-antibody conjugate can be administered to the patient in an aqueoussolution containing one or more pharmaceutically acceptable excipients,such as a viscosity-modifying agent. The aqueous solution may besterilized using techniques described herein or known in the art. Theantibody, antigen-binding fragment thereof, or drug-antibody conjugatecan be administered to the patient at a dosage of, for example, from0.001 mg/kg to 100 mg/kg prior to administration of a hematopoietic stemcell graft to the patient. The antibody, antigen-binding fragmentthereof, or drug-antibody conjugate can be administered to the patientat a time that optimally promotes engraftment of the exogenoushematopoietic stem cells, for instance, from 1 hour to 1 week (e.g., 1hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days) ormore prior to administration of the exogenous hematopoietic stem celltransplant.

Following the conclusion of conditioning therapy, the patient may thenreceive an infusion (e.g., an intravenous infusion) of exogenoushematopoietic stem cells, such as from the same physician that performedthe conditioning therapy or from a different physician. The physicianmay administer the patient an infusion of autologous, syngeneic, orallogeneic hematopoietic stem cells, for instance, at a dosage of from1×10³ to 1×10⁹ hematopoietic stem cells/kg. The physician may monitorthe engraftment of the hematopoietic stem cell transplant, for example,by withdrawing a blood sample from the patient and determining theincrease in concentration of hematopoietic stem cells or cells of thehematopoietic lineage (such as megakaryocytes, thrombocytes, platelets,erythrocytes, mast cells, myeoblasts, basophils, neutrophils,eosinophils, microglia, granulocytes, monocytes, osteoclasts,antigen-presenting cells, macrophages, dendritic cells, natural killercells, T-lymphocytes, and B-lymphocytes) following administration of thetransplant. This analysis may be conducted, for example, from 1 hour to6 months, or more, following hematopoietic stem cell transplant therapy(e.g., 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22hours, 23 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23weeks, 24 weeks, or more). A finding that the concentration ofhematopoietic stem cells or cells of the hematopoietic lineage hasincreased (e.g., by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, or more) following thetransplant therapy relative to the concentration of the correspondingcell type prior to transplant therapy provides one indication thattreatment with the anti-CD90 antibody, antigen-binding fragment thereof,or drug-antibody conjugate has successfully promoted engraftment of thetransplanted hematopoietic stem cell graft.

Example 5. Administration of an Anti-CD110 Antibody to a Human Patientin Preparation for Hematopoietic Stem Cell Transplant Therapy

Using the methods disclosed herein, a physician of skill in the art canadminister to a human patient in need of hematopoietic stem celltransplant therapy an antibody or antigen-binding fragment thereofcapable of binding an antigen expressed by hematopoietic stem cells,such as an antibody or antigen-biding fragment thereof that binds CD110.In this way, a population of endogenous hematopoietic stem cells can bedepleted prior to administration of an exogenous hematopoietic stem cellgraft so as to promote engraftment of the hematopoietic stem cell graft.The antibody may be covalently conjugated to a toxin, such as acytotoxic molecule described herein or known in the art. For instance,an anti-CD110 antibody or antigen-binding fragment thereof can becovalently conjugated to a cytotoxin, such as pseudomonas exotoxin A,deBouganin, diphtheria toxin, an amatoxin, such as α-amanitin, saporin,maytansine, a maytansinoid, an auristatin, an anthracycline, acalicheamicin, irinotecan, SN-38, a duocarmycin, apyrrolobenzodiazepine, a pyrrolobenzodiazepine dimer, anindolinobenzodiazepine, an indolinobenzodiazepine dimer, or a variantthereof. This conjugation can be performed using covalent bond-formingtechniques described herein or known in the art. The antibody,antigen-binding fragment thereof, or drug-antibody conjugate cansubsequently be administered to the patient, for example, by intravenousadministration, prior to transplantation of exogenous hematopoietic stemcells (such as autologous, syngeneic, or allogeneic hematopoietic stemcells) to the patient.

The anti-CD110 antibody, antigen-binding fragment thereof, ordrug-antibody conjugate can be administered in an amount sufficient toreduce the quantity of endogenous hematopoietic stem cells, for example,by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more prior tohematopoietic stem cell transplant therapy. The reduction inhematopoietic stem cell count can be monitored using conventionaltechniques known in the art, such as by FACS analysis of cellsexpressing characteristic hematopoietic stem cell surface antigens in ablood sample withdrawn from the patient at varying intervals duringconditioning therapy. For instance, a physician of skill in the art canwithdraw a blood sample from the patient at various time points duringconditioning therapy and determine the extent of endogenoushematopoietic stem cell reduction by conducting a FACS analysis toelucidate the relative concentrations of hematopoietic stem cells in thesample using antibodies that bind to hematopoietic stem cell markerantigens. According to some embodiments, when the concentration ofhematopoietic stem cells has reached a minimum value in response toconditioning therapy with an anti-CD135 antibody, antigen-bindingfragment thereof, or drug-antibody conjugate, the physician may concludethe conditioning therapy, and may begin preparing the patient forhematopoietic stem cell transplant therapy.

The anti-CD110 antibody, antigen-binding fragment thereof, ordrug-antibody conjugate can be administered to the patient in an aqueoussolution containing one or more pharmaceutically acceptable excipients,such as a viscosity-modifying agent. The aqueous solution may besterilized using techniques described herein or known in the art. Theantibody, antigen-binding fragment thereof, or drug-antibody conjugatecan be administered to the patient at a dosage of, for example, from0.001 mg/kg to 100 mg/kg prior to administration of a hematopoietic stemcell graft to the patient. The antibody, antigen-binding fragmentthereof, or drug-antibody conjugate can be administered to the patientat a time that optimally promotes engraftment of the exogenoushematopoietic stem cells, for instance, from 1 hour to 1 week (e.g., 1hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days) ormore prior to administration of the exogenous hematopoietic stem celltransplant.

Following the conclusion of conditioning therapy, the patient may thenreceive an infusion (e.g., an intravenous infusion) of exogenoushematopoietic stem cells, such as from the same physician that performedthe conditioning therapy or from a different physician. The physicianmay administer the patient an infusion of autologous, syngeneic, orallogeneic hematopoietic stem cells, for instance, at a dosage of from1×10³ to 1×10⁹ hematopoietic stem cells/kg. The physician may monitorthe engraftment of the hematopoietic stem cell transplant, for example,by withdrawing a blood sample from the patient and determining theincrease in concentration of hematopoietic stem cells or cells of thehematopoietic lineage (such as megakaryocytes, thrombocytes, platelets,erythrocytes, mast cells, myeoblasts, basophils, neutrophils,eosinophils, microglia, granulocytes, monocytes, osteoclasts,antigen-presenting cells, macrophages, dendritic cells, natural killercells, T-lymphocytes, and B-lymphocytes) following administration of thetransplant. This analysis may be conducted, for example, from 1 hour to6 months, or more, following hematopoietic stem cell transplant therapy(e.g., 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22hours, 23 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23weeks, 24 weeks, or more). A finding that the concentration ofhematopoietic stem cells or cells of the hematopoietic lineage hasincreased (e.g., by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, or more) following thetransplant therapy relative to the concentration of the correspondingcell type prior to transplant therapy provides one indication thattreatment with the anti-CD110 antibody, antigen-binding fragmentthereof, or drug-antibody conjugate has successfully promotedengraftment of the transplanted hematopoietic stem cell graft.

Example 6. Generating Antibodies Capable of Binding Hematopoietic StemCells by Phage Display

An exemplary method for in vitro evolution of anti-CD45 (e.g.,anti-CD45RO), anti-CD135, anti-CD34, anti-CD90, or anti-CD110 antibodiesfor use with the compositions and methods described herein is phagedisplay. Phage display libraries can be created by making a designedseries of mutations or variations within a coding sequence for the CDRsof an antibody or the analogous regions of an antibody-like scaffold(e.g., the BC, CD, and DE loops of ¹⁰Fn3 domains). The templateantibody-encoding sequence into which these mutations are introduced maybe, for example, a naive human germline sequence. These mutations can beperformed using standard mutagenesis techniques known in the art. Eachmutant sequence thus encodes an antibody corresponding to the templatesave for one or more amino acid variations. Retroviral and phage displayvectors can be engineered using standard vector construction techniquesknown in the art. P3 phage display vectors along with compatible proteinexpression vectors can be used to generate phage display vectors forantibody diversification.

The mutated DNA provides sequence diversity, and each transformant phagedisplays one variant of the initial template amino acid sequence encodedby the DNA, leading to a phage population (library) displaying a vastnumber of different but structurally related amino acid sequences. Dueto the well-defined structure of antibody hypervariable regions, theamino acid variations introduced in a phage display screen are expectedto alter the binding properties of the binding peptide or domain withoutsignificantly altering its overall molecular structure.

In a typical screen, a phage library may be contacted with and allowedto bind one of the foregoing antigens or an epitope thereof. Tofacilitate separation of binders and non-binders, it is convenient toimmobilize the target on a solid support. Phage bearing a CD45−, CD135−,CD34−, CD90−, or CD110-binding moiety can form a complex with the targeton the solid support, whereas non-binding phage remain in solution andcan be washed away with excess buffer. Bound phage can then liberatedfrom the target by changing the buffer to an extreme pH (pH 2 or pH 10),changing the ionic strength of the buffer, adding denaturants, or otherknown means.

The recovered phage can then be amplified through infection of bacterialcells, and the screening process can be repeated with the new pool thatis now depleted in non-binding antibodies and enriched for antibodiesthat bind CD45 (e.g., CD45RO), CD135, CD34, CD90, or CD110. The recoveryof even a few binding phage is sufficient to amplify the phage for asubsequent iteration of screening. After a few rounds of selection, thegene sequences encoding the antibodies or antigen-binding fragmentsthereof derived from selected phage clones in the binding pool aredetermined by conventional methods, thus revealing the peptide sequencethat imparts binding affinity of the phage to the target. During thepanning process, the sequence diversity of the population diminisheswith each round of selection until desirable peptide-binding antibodiesremain. The sequences may converge on a small number of relatedantibodies or antigen-binding fragments thereof. An increase in thenumber of phage recovered at each round of selection is an indicationthat convergence of the library has occurred in a screen.

Example 7. Producing Humanized Antibodies that Bind a Hematopoietic StemCell Antigen

Non-human antibodies that bind CD45 (e.g., CD45RO), CD135, CD34, CD90,or CD110 can be humanized, for instance, according to the followingprocedure. Consensus human antibody heavy chain and light chainsequences are known in the art (see e.g., the “VBASE” human germlinesequence database; Kabat et al. Sequences of Proteins of ImmunologicalInterest, Fifth Edition, U.S. Department of Health and Human Services,NIH Publication No. 91-3242, 1991; Tomlinson et al., J. Mol. Biol.227:776-798, 1992; and Cox et al. Eur. J. Immunol. 24:827-836, 1994, thedisclosures of each of which are incorporated herein by reference asthey pertain to consensus human antibody heavy chain and light chainsequences. Using established procedures, one of skill in the art canidentify the variable domain framework residues and CDRs of a consensusantibody sequence (e.g., by sequence alignment). One can substitute oneor more CDRs of the heavy chain and/or light chain variable domains ofconsensus human antibody with one or more corresponding CDRs of anon-human antibody that binds CD45 (e.g., CD45RO), CD135, CD34, CD90, orCD110 as described herein in order to produce a humanized antibody. ThisCDR exchange can be performed using gene editing techniques describedherein or known in the art.

One example of a variable domain of a consensus human antibody containsthe heavy chain variable domainEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYAMSWVRQAPGKGLEWVAVISENGSDTYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCARDRGGAVSYFDVWGQGTLVTVSS (SEQ ID NO: 34)and the light chain variable domainDIQMTQSPSSLSASVGDRVTITCRASQDVSSYLAWYQQKPGKAPKLLIYAASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSLPYTFGQGTKVEIKRT (SEQ ID NO: 35), identified inU.S. Pat. No. 6,054,297, the disclosure of which is incorporated hereinby reference as it pertains to human antibody consensus sequences. TheCDRs in the above sequences are shown in bold.

To produce humanized antibodies, one can recombinantly express apolynucleotide encoding the above consensus sequence in which one ormore variable region CDRs have been replaced with one or more variableregion CDR sequences of a non-human antibody that binds CD45 (e.g.,CD45RO), CD135, CD34, CD90, or CD110. As the affinity of the antibodyfor the hematopoietic stem cell antigen is determined primarily by theCDR sequences, the resulting humanized antibody is expected to exhibitan affinity for the hematopoietic stem cell antigen that is about thesame as that of the non-human antibody from which the humanized antibodywas derived. Methods of determining the affinity of an antibody for atarget antigen include, for instance, ELISA-based techniques describedherein and known in the art, as well as surface plasmon resonance,fluorescence anisotropy, and isothermal titration calorimetry, amongothers.

Example 8. Ability of Anti-CD45 Antibody-Drug Conjugates to DepletePopulations of CD45+ Cells

To investigate the ability of anti-CD45 antibody-drug conjugates to killCD45+ Reh cells, a series of anti-CD45 monoclonal antibodies of variousisotypes, Ab1, Ab2, and Ab3, were bound to saporin-conjugated Fabfragments and were subsequently incubated with CD45+ Reh cells for threedays. As a negative control, an isotype-matched antibody-saporinconjugate was generated for each of Ab1, Ab2, and Ab3, and these werewas incubated with CD45+ Reh cells for the same three-day period. Cellviability was then assessed using the CellTiter-Glo™ assay. As shown inFIG. 1, the various antibody-saporin conjugates were capable of killingCD45+ Reh cells with different potencies, and each was capable ofdepleting the CD45+ Reh cell line in a dose-dependent fashion.

To investigate the ability of anti-CD45 monoclonal antibody-saporinconjugates to deplete human CD34+ hematopoietic stem cells in vitro, ahuman IgG1 anti-CD45 monoclonal antibody-saporin conjugate orisotype-matched control was incubated with CD34+ cells at variousconcentrations. Cell viability was subsequently assessed following asix-day incubation period using flow cytometry. The results of thisexperiment are reported in FIG. 2. As shown therein, anti-CD45antibody-saporin conjugates were found to be capable of depleting humanCD34+ hematopoietic stem cells in a dose-dependent manner.

Taken together, these results demonstrate that anti-CD45 antibodiesconjugated to saporin can be used to deplete a population ofhematopoietic stem cells in a subject, for instance, in preparation forhematopoietic stem cell transplant therapy, so as to provide a niche forto which the hematopoietic stem cells may home.

Other Embodiments

All publications, patents, and patent applications mentioned in thisspecification are incorporated herein by reference to the same extent asif each independent publication or patent application was specificallyand individually indicated to be incorporated by reference.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from theinvention that come within known or customary practice within the art towhich the invention pertains and may be applied to the essentialfeatures hereinbefore set forth, and follows in the scope of the claims.

Other embodiments are within the claims.

What is claimed is:
 1. A method of depleting a population of CD45RO+cells in a human patient, the method comprising administering to thepatient an effective amount of an antibody or antigen-binding fragmentthereof capable of binding CD45RO.
 2. A method of depleting a populationof CD45RO+ cells in a human patient in need of a hematopoietic stem celltransplant, the method comprising administering to the patient aneffective amount of an antibody or antigen-binding fragment thereofcapable of binding CD45RO prior to the patient receiving a transplantcomprising hematopoietic stem cells.
 3. A method comprisingadministering to a human patient a transplant comprising hematopoieticstem cells, wherein the patient has been previously administered anantibody or antigen-binding fragment thereof capable of binding CD45ROin an amount sufficient to deplete a population of CD45RO+ cells in thepatient.
 4. A method comprising: a. administering to a human patient anantibody or antigen-binding fragment thereof capable of binding CD45ROin an amount sufficient to deplete a population of CD45RO+ cells in thepatient; and b. subsequently administering to the patient a transplantcomprising hematopoietic stem cells.
 5. The method of any one of claims1-4, wherein the antibody or antigen-binding fragment thereof isconjugated to a cytotoxin.
 6. A method of depleting a population ofCD135+ cells in a human patient, the method comprising administering tothe patient an effective amount of an antibody or antigen-bindingfragment thereof capable of binding CD135, wherein the antibody orantigen-binding fragment thereof is conjugated to a cytotoxin.
 7. Amethod of depleting a population of CD135+ cells in a human patient inneed of a hematopoietic stem cell transplant, the method comprisingadministering to the patient an effective amount of an antibody orantigen-binding fragment thereof capable of binding CD135 prior to thepatient receiving a transplant comprising hematopoietic stem cells,wherein the antibody or antigen-binding fragment thereof is conjugatedto a cytotoxin.
 8. A method comprising administering to a human patienta transplant comprising hematopoietic stem cells, wherein the patienthas been previously administered an antibody or antigen-binding fragmentthereof capable of binding CD135 in an amount sufficient to deplete apopulation of CD135+ cells in the patient, wherein the antibody orantigen-binding fragment thereof is conjugated to a cytotoxin.
 9. Amethod comprising: a. administering to a human patient an antibody orantigen-binding fragment thereof capable of binding CD135 in an amountsufficient to deplete a population of CD135+ cells in the patient; andb. subsequently administering to the patient a transplant comprisinghematopoietic stem cells, wherein the antibody or antigen-bindingfragment thereof is conjugated to a cytotoxin.
 10. The method of any oneof claims 6-9, wherein the antibody or antigen-binding fragment thereofcomprises the following complementarity determining regions (CDRs):(SEQ ID NO: 1) a. a CDR-H1 having the amino acid sequence SYYMH;(SEQ ID NO: 2) b. a CDR-H2 having the amino acid sequenceIINPSGGSTSYAQKFQG; (SEQ ID NO: 3)c. a CDR-H3 having the amino acid sequence GVGAHDAFDI or (SEQ ID NO: 4)VVAAAVADY; (SEQ ID NO: 5) d. a CDR-L1 having the amino acid sequenceRSSQSLLHSNGNNYLD or (SEQ ID NO: 6) RSSQSLLHSNGYNYLD; (SEQ ID NO: 7)e. a CDR-L2 having the amino acid sequence LGSNRAS; and (SEQ ID NO: 8)f. a CDR-L3 having the amino acid sequence MQGTHPAIS or (SEQ ID NO: 9)MQSLQTPFT.


11. The method of any one of claims 6-9, wherein the antibody orantigen-binding fragment thereof comprises the following CDRs:(SEQ ID NO: 10) a. a CDR-H1 having the amino acid sequence SYAIS;(SEQ ID NO: 11) b. a CDR-H2 having the amino acid sequenceGIIPIFGTANYAQKFQG; (SEQ ID NO: 12)c. a CDR-H3 having the amino acid sequence FALFGFREQAFDI;(SEQ ID NO: 13) d. a CDR-L1 having the amino acid sequence RASQSISSYLN;(SEQ ID NO: 14) e. a CDR-L2 having the amino acid sequence AASSLQS; and(SEQ ID NO: 15) f. a CDR-L3 having the amino acid sequence QQSYSTPFT.


12. A method of depleting a population of CD135+ cells in a humanpatient, the method comprising administering to the patient an effectiveamount of human Flt3 ligand, or a fragment thereof capable of bindingCD135.
 13. A method of depleting a population of CD135+ cells in a humanpatient in need of a hematopoietic stem cell transplant, the methodcomprising administering to the patient an effective amount of humanFlt3 ligand, or a fragment thereof capable of binding CD135, prior tothe patient receiving a transplant comprising hematopoietic stem cells.14. A method comprising administering to a human patient a transplantcomprising hematopoietic stem cells, wherein the patient has beenpreviously administered human Flt3 ligand, or a fragment thereof capableof binding CD135, in an amount sufficient to deplete a population ofCD135+ cells in the patient.
 15. A method comprising: a. administeringto a human patient human Flt3 ligand, or a fragment thereof capable ofbinding CD135, in an amount sufficient to deplete a population of CD135+cells in the patient; and b. subsequently administering to the patient atransplant comprising hematopoietic stem cells.
 16. The method of anyone of claims 12-15, wherein the human Flt3 ligand or fragment thereofis covalently bound to an Fc domain.
 17. The method of claim 16, whereinthe N-terminus of the human Flt3 ligand or fragment thereof iscovalently bound to the Fc domain.
 18. The method of claim 16, whereinthe C-terminus of the human Flt3 ligand or fragment thereof iscovalently bound to the Fc domain.
 19. The method of any one of claims16-18, wherein the Fc domain is covalently bound to a cytotoxin.
 20. Themethod of any one of claims 12-15, wherein the human Flt3 ligand orfragment thereof is covalently bound to a cytotoxin.
 21. The method ofclaim 20, wherein the N-terminus of the human Flt3 ligand or fragmentthereof is covalently bound to the cytotoxin.
 22. The method of claim20, wherein the C-terminus of the human Flt3 ligand or fragment thereofis covalently bound to the cytotoxin.
 23. The method of any one ofclaims 20-22, wherein the cytotoxin is covalently bound to an Fc domain.24. The method of any claim 20, wherein the human Flt3 ligand orfragment thereof is covalently bound to the cytotoxin at one site and iscovalently bound to an Fc domain at a different site.
 25. The method ofany one of claims 16-19, 23, and 24, wherein the Fc domain is a humanIgG1, IgG2, IgG3, or IgG4 isotype Fc domain.
 26. A method of depleting apopulation of CD34+ cells in a human patient, the method comprisingadministering to the patient an effective amount of an antibody orantigen-binding fragment thereof capable of binding CD34, wherein theantibody or antigen-binding fragment thereof is conjugated to acytotoxin.
 27. A method of depleting a population of CD34+ cells in ahuman patient in need of a hematopoietic stem cell transplant, themethod comprising administering to the patient an effective amount of anantibody or antigen-binding fragment thereof capable of binding CD34prior to the patient receiving a transplant comprising hematopoieticstem cells, wherein the antibody or antigen-binding fragment thereof isconjugated to a cytotoxin.
 28. A method comprising administering to ahuman patient a transplant comprising hematopoietic stem cells, whereinthe patient has been previously administered an antibody orantigen-binding fragment thereof capable of binding CD34 in an amountsufficient to deplete a population of CD34+ cells in the patient,wherein the antibody or antigen-binding fragment thereof is conjugatedto a cytotoxin.
 29. A method comprising: a. administering to a humanpatient an antibody or antigen-binding fragment thereof capable ofbinding CD34 in an amount sufficient to deplete a population of CD34+cells in the patient; and b. subsequently administering to the patient atransplant comprising hematopoietic stem cells, wherein the antibody orantigen-binding fragment thereof is conjugated to a cytotoxin.
 30. Amethod of depleting a population of CD90+ cells in a human patient, themethod comprising administering to the patient an effective amount of anantibody or antigen-binding fragment thereof capable of binding CD90,wherein the antibody or antigen-binding fragment thereof is conjugatedto a cytotoxin.
 31. A method of depleting a population of CD90+ cells ina human patient in need of a hematopoietic stem cell transplant, themethod comprising administering to the patient an effective amount of anantibody or antigen-binding fragment thereof capable of binding CD90prior to the patient receiving a transplant comprising hematopoieticstem cells, wherein the antibody or antigen-binding fragment thereof isconjugated to a cytotoxin.
 32. A method comprising administering to ahuman patient a transplant comprising hematopoietic stem cells, whereinthe patient has been previously administered an antibody orantigen-binding fragment thereof capable of binding CD90 in an amountsufficient to deplete a population of CD90+ cells in the patient,wherein the antibody or antigen-binding fragment thereof is conjugatedto a cytotoxin.
 33. A method comprising: a. administering to a humanpatient an antibody or antigen-binding fragment thereof capable ofbinding CD90 in an amount sufficient to deplete a population of CD90+cells in the patient; and b. subsequently administering to the patient atransplant comprising hematopoietic stem cells, wherein the antibody orantigen-binding fragment thereof is conjugated to a cytotoxin.
 34. Amethod of depleting a population of CD110+ cells in a human patient, themethod comprising administering to the patient an effective amount of anantibody or antigen-binding fragment thereof capable of binding CD110,wherein the antibody or antigen-binding fragment thereof is conjugatedto a cytotoxin.
 35. A method of depleting a population of CD110+ cellsin a human patient in need of a hematopoietic stem cell transplant, themethod comprising administering to the patient an effective amount of anantibody or antigen-binding fragment thereof capable of binding CD110prior to the patient receiving a transplant comprising hematopoieticstem cells, wherein the antibody or antigen-binding fragment thereof isconjugated to a cytotoxin.
 36. A method comprising administering to ahuman patient a transplant comprising hematopoietic stem cells, whereinthe patient has been previously administered an antibody orantigen-binding fragment thereof capable of binding CD110 in an amountsufficient to deplete a population of CD110+ cells in the patient,wherein the antibody or antigen-binding fragment thereof is conjugatedto a cytotoxin.
 37. A method comprising: a. administering to a humanpatient an antibody or antigen-binding fragment thereof capable ofbinding CD110 in an amount sufficient to deplete a population of CD110+cells in the patient; and b. subsequently administering to the patient atransplant comprising hematopoietic stem cells, wherein the antibody orantigen-binding fragment thereof is conjugated to a cytotoxin.
 38. Themethod of any one of claims 5-11 and 19-37, wherein the cytotoxin isselected from the group consisting of an amatoxin, pseudomonas exotoxinA, deBouganin, diphtheria toxin, saporin, maytansine, a maytansinoid, anauristatin, an anthracycline, a calicheamicin, irinotecan, SN-38, aduocarmycin, a pyrrolobenzodiazepine, a pyrrolobenzodiazepine dimer, anindolinobenzodiazepine, and an indolinobenzodiazepine dimer, or avariant thereof.
 39. A method of depleting a population of CD45+ cellsin a human patient, the method comprising administering to the patientan effective amount of an antibody or antigen-binding fragment thereofcapable of binding CD45, wherein the antibody or antigen-bindingfragment thereof is conjugated to a cytotoxin selected from the groupconsisting of an amatoxin, pseudomonas exotoxin A, deBouganin,diphtheria toxin, saporin, maytansine, a maytansinoid, an auristatin, ananthracycline, a calicheamicin, irinotecan, SN-38, a duocarmycin, apyrrolobenzodiazepine, a pyrrolobenzodiazepine dimer, anindolinobenzodiazepine, and an indolinobenzodiazepine dimer, or avariant thereof.
 40. A method of depleting a population of CD45+ cellsin a human patient in need of a hematopoietic stem cell transplant, themethod comprising administering to the patient an effective amount of anantibody or antigen-binding fragment thereof capable of binding CD45prior to the patient receiving a transplant comprising hematopoieticstem cells, wherein the antibody or antigen-binding fragment thereof isconjugated to a cytotoxin selected from the group consisting of anamatoxin, pseudomonas exotoxin A, deBouganin, diphtheria toxin, saporin,maytansine, a maytansinoid, an auristatin, an anthracycline, acalicheamicin, irinotecan, SN-38, a duocarmycin, apyrrolobenzodiazepine, a pyrrolobenzodiazepine dimer, anindolinobenzodiazepine, and an indolinobenzodiazepine dimer, or avariant thereof.
 41. A method of comprising administering to a humanpatient a transplant comprising hematopoietic stem cells, wherein thepatient has been previously administered an antibody or antigen-bindingfragment thereof capable of binding CD45 in an amount sufficient todeplete a population of CD45+ cells in the patient, wherein the antibodyor antigen-binding fragment thereof is conjugated to a cytotoxinselected from the group consisting of an amatoxin, pseudomonas exotoxinA, deBouganin, diphtheria toxin, saporin, maytansine, a maytansinoid, anauristatin, an anthracycline, a calicheamicin, irinotecan, SN-38, aduocarmycin, a pyrrolobenzodiazepine, a pyrrolobenzodiazepine dimer, anindolinobenzodiazepine, and an indolinobenzodiazepine dimer, or avariant thereof.
 42. A method comprising: a. administering to a humanpatient an antibody or antigen-binding fragment thereof capable ofbinding CD45 in an amount sufficient to deplete a population of CD45+cells in the patient; and b. subsequently administering to the patient atransplant comprising hematopoietic stem cells, wherein the antibody orantigen-binding fragment thereof is conjugated to a cytotoxin selectedfrom the group consisting of an amatoxin, pseudomonas exotoxin A,deBouganin, diphtheria toxin, saporin, maytansine, a maytansinoid, anauristatin, an anthracycline, a calicheamicin, irinotecan, SN-38, aduocarmycin, a pyrrolobenzodiazepine, a pyrrolobenzodiazepine dimer, anindolinobenzodiazepine, and an indolinobenzodiazepine dimer, or avariant thereof.
 43. The method of any one of claims 5-11 and 19-42,wherein the antibody, antigen-binding fragment thereof, or ligandconjugated to a cytotoxin is represented by the formula Ab-Am, whereinAb is the antibody, antigen-binding fragment thereof, or ligand and Amis an amatoxin represented by formula (I)

wherein R₁ is H, OH, OR_(A), or OR_(C); R₂ is H, OH, OR_(B), or OR_(C);R_(A) and R_(B), together with the oxygen atoms to which they are bound,combine to form an optionally substituted 5-membered heterocyclolalkylgroup; R₃ is H, R_(C), or R_(D); R₄, R₅, R₆, and R₇ are eachindependently H, OH, OR_(C), OR_(D), R_(C), or R_(D); R₈ is OH, NH₂,OR_(C), OR_(D), NHR_(C), or NR_(C)R_(D); R₉ is H, OH, OR_(C), or OR_(D);X is —S—, —S(O)—, or —SO₂—; R_(C) is -L-Z; R_(D) is optionallysubstituted C₁-C₆ alkyl, optionally substituted C₁-C₆ heteroalkyl,optionally substituted C₂-C₆ alkenyl, optionally substituted C₂-C₆heteroalkenyl, optionally substituted C₂-C₆ alkynyl, optionallysubstituted C₂-C₆ heteroalkynyl, optionally substituted cycloalkyl,optionally substituted heterocycloalkyl, optionally substituted aryl, oroptionally substituted heteroaryl; L is optionally substituted C₁-C₆alkylene, optionally substituted C₁-C₆ heteroalkylene, optionallysubstituted C₂-C₆ alkenylene, optionally substituted C₂-C₆heteroalkenylene, optionally substituted C₂-C₆ alkynylene, optionallysubstituted C₂-C₆ heteroalkynylene, optionally substitutedcycloalkylene, optionally substituted heterocycloalkylene, optionallysubstituted arylene, or optionally substituted heteroarylene; and Z is achemical moiety formed from a coupling reaction between a reactivesubstituent present on L and a reactive substituent present within theantibody or antigen-binding fragment thereof, wherein Am comprisesexactly one R_(C) substituent.
 44. The method of claim 43, wherein Am isan amatoxin represented by formula (IA).


45. The method of claim 43, wherein Am is an amatoxin represented byformula (IB).


46. The method of claim 44 or 45, wherein R_(A) and R_(B), together withthe oxygen atoms to which they are bound, combine to form:

wherein Y is selected from O, S, NR_(E), and CR_(E)R_(E′), and R_(E) andR_(E′) are each independently optionally substituted C₁-C₆alkylene-R_(C), optionally substituted C₁-C₆ heteroalkylene-R_(C),optionally substituted C₂-C₆ alkenylene-R_(C), optionally substitutedC₂-C₆ heteroalkenylene-R_(C), optionally substituted C₂-C₆alkynylene-R_(C), optionally substituted C₂-C₆ heteroalkynylene-R_(C),optionally substituted cycloalkylene-R_(C), optionally substitutedheterocycloalkylene-R_(C), optionally substituted arylene-R_(C), oroptionally substituted heteroarylene-R_(C).
 47. The method of claim 46,wherein R_(A) and R_(B), together with the oxygen atoms to which theyare bound, combine to form:


48. The method of claim 44 or 45, wherein R₁ is H, OH, or OR_(A); R₂ isH, OH, or OR_(B); R_(A) and R_(B), together with the oxygen atoms towhich they are bound, combine to form:

R₃, R₄, R₆, and R₇ are each H; R₅ is OR_(C); R₅ is OH or NH₂; and R₉ isH or OH.
 49. The method of claim 44 or 45, wherein R₁ and R₂ are eachindependently H or OH; R₃ is R_(C); R₄, R₆, and R₇ are each H; R₅ is H,OH, or OC₁-C₆ alkyl; R₅ is OH or NH₂; and R₉ is H or OH.
 50. The methodof claim 44 or 45, wherein R₁ and R₂ are each independently H or OH; R₃,R₆, and R₇ are each H; R₄ and R₅ are each independently H, OH, OR_(C),or R_(C); R₅ is OH or NH₂; and R₉ is H or OH.
 51. The method of claim 44or 45, wherein R₁ and R₂ are each independently H or OH; R₃, R₆, and R₇are each H; R₄ and R₅ are each independently H or OH; R₈ is OR_(C) orNHR_(C); and R₉ is H or OH.
 52. The method of any one of claims 5-11 and19-42, wherein the antibody, antigen-binding fragment thereof, or ligandconjugated to a cytotoxin is represented by the formula Ab-Am, whereinAb is the antibody, antigen-binding fragment thereof, or ligand and Amis an amatoxin represented by formula (II)

wherein X is S, SO, or SO₂; R₁ is H or a linker covalently bound to theantibody or antigen-binding fragment thereof; and R₂ is H or a linkercovalently bound to the antibody or antigen-binding fragment thereof;wherein when R₁ is H, R₂ is the linker, and when R₂ is H, R₁ is thelinker.
 53. The method of any one of claims 5-11 and 19-42, wherein thecytotoxin is a maytansinoid selected from the group consisting of DM1and DM4.
 54. The method of any one of claims 5-11 and 19-42, wherein thecytotoxin is an auristatin selected from the group consisting ofmonomethyl auristatin E and monomethyl auristatin F.
 55. The method ofany one of claims 5-11 and 19-42, wherein the cytotoxin is ananthracycline selected from the group consisting of daunorubicin,doxorubicin, epirubicin, and idarubicin.
 56. The method of any one ofclaims 1-11 and 26-55, wherein the antibody or antigen-binding fragmentthereof is selected from the group consisting of a monoclonal antibodyor antigen-binding fragment thereof, a polyclonal antibody orantigen-binding fragment thereof, a humanized antibody orantigen-binding fragment thereof, a bispecific antibody orantigen-binding fragment thereof, a dual-variable immunoglobulin domain,a single-chain Fv molecule (scFv), a diabody, a triabody, a nanobody, anantibody-like protein scaffold, a Fv fragment, a Fab fragment, a F(ab′)₂molecule, and a tandem di-scFv.
 57. The method of any one of claims 1-11and 26-56, wherein the antibody has an isotype selected from the groupconsisting of IgG, IgA, IgM, IgD, and IgE.
 58. The method of any one ofclaims 1-57, wherein the antibody, antigen-binding fragment thereof, orligand is internalized by a cancer cell, autoimmune cell, orhematopoietic stem cell following administration to the patient.
 59. Themethod of any one of claims 1-58, wherein the antibody, antigen-bindingfragment thereof, or ligand is capable of promoting necrosis of a cancercell, autoimmune cell, or hematopoietic stem cell.
 60. The method of anyone of claims 2-5, 7-11, 13-25, 27-29, 31-33, 35-38, and 40-59, whereinthe antibody, antigen-binding fragment thereof, or ligand is capable ofrecruiting one or more complement proteins to the hematopoietic stemcell upon administration to the patient.
 61. The method of any one ofclaims 2-5, 7-11, 13-25, 27-29, 31-33, 35-38, and 40-60, wherein thetransplant comprising hematopoietic stem cells is administered to thepatient after the concentration of the antibody, antigen-bindingfragment thereof, or ligand has substantially cleared from the blood ofthe patient.
 62. The method of any one of claims 2-5, 7-11, 13-25,27-29, 31-33, 35-38, and 40-61, wherein the hematopoietic stem cells orprogeny thereof maintain hematopoietic stem cell functional potentialafter two or more days following transplantation of the hematopoieticstem cells into the patient.
 63. The method of any one of claims 2-5,7-11, 13-25, 27-29, 31-33, 35-38, and 40-62, wherein the hematopoieticstem cells or progeny thereof are capable of localizing to hematopoietictissue and/or reestablishing hematopoiesis following transplantation ofthe hematopoietic stem cells into the patient.
 64. The method of any oneof claims 2-5, 7-11, 13-25, 27-29, 31-33, 35-38, and 40-63, wherein upontransplantation into the patient, the hematopoietic stem cells give riseto recovery of a population of cells selected from the group consistingof megakaryocytes, thrombocytes, platelets, erythrocytes, mast cells,myeoblasts, basophils, neutrophils, eosinophils, microglia,granulocytes, monocytes, osteoclasts, antigen-presenting cells,macrophages, dendritic cells, natural killer cells, T-lymphocytes, andB-lymphocytes.
 65. The method of any one of claims 1-64, wherein thepatient is suffering from a stem cell disorder.
 66. The method of anyone of claims 1-65, wherein the patient is suffering from ahemoglobinopathy disorder.
 67. The method of claim 66, wherein thehemoglobinopathy disorder is selected from the group consisting ofsickle cell anemia, thalassemia, Fanconi anemia, aplastic anemia, andWiskott-Aldrich syndrome.
 68. The method of claim 66, wherein thehemoglobinopathy disorder is Fanconi anemia.
 69. The method of claim 66,wherein the hemoglobinopathy disorder is aplastic anemia.
 70. The methodof claim 66, wherein the hemoglobinopathy disorder is sickle cellanemia.
 71. The method of claim 66, wherein the hemoglobinopathydisorder is thalassemia.
 72. The method of any one of claims 1-71,wherein the patient is suffering from a myelodysplastic disorder. 73.The method of any one of claims 1-72, wherein the patient is sufferingfrom an immunodeficiency disorder.
 74. The method of claim 73, whereinthe immunodeficiency disorder is a congenital immunodeficiency.
 75. Themethod of claim 73, wherein the immunodeficiency disorder is an acquiredimmunodeficiency.
 76. The method of claim 75, wherein the acquiredimmunodeficiency is human immunodeficiency virus or acquired immunedeficiency syndrome.
 77. The method of any one of claims 1-76, whereinthe patient is suffering from a metabolic disorder.
 78. The method ofclaim 77, wherein the metabolic disorder is selected from the groupconsisting of glycogen storage diseases, mucopolysaccharidoses,Gaucher's Disease, Hurlers Disease, sphingolipidoses, and metachromaticleukodystrophy.
 79. The method of any one of claims 1-78, wherein thepatient is suffering from cancer.
 80. The method of claim 79, whereinthe cancer is selected from the group consisting of leukemia, lymphoma,multiple myeloma, and neuroblastoma.
 81. The method of claim 79, whereinthe cancer is a hematological cancer.
 82. The method of claim 79,wherein the cancer is acute myeloid leukemia.
 83. The method of claim79, wherein the cancer is acute lymphoid leukemia.
 84. The method ofclaim 79, wherein the cancer is chronic myeloid leukemia.
 85. The methodof claim 79, wherein the cancer is chronic lymphoid leukemia.
 86. Themethod of claim 79, wherein the cancer is diffuse large B-cell lymphoma.87. The method of claim 79, wherein the cancer is non-Hodgkin'slymphoma.
 88. The method of any one of claims 1-87, wherein the patientis suffering from a disorder selected from the group consisting ofadenosine deaminase deficiency and severe combined immunodeficiency,hyper immunoglobulin M syndrome, Chediak-Higashi disease, hereditarylymphohistiocytosis, osteopetrosis, osteogenesis imperfecta, storagediseases, thalassemia major, systemic sclerosis, systemic lupuserythematosus, multiple sclerosis, and juvenile rheumatoid arthritis.89. The method of any one of claims 1-88, wherein the patient issuffering from an autoimmune disorder.
 90. The method of claim 89,wherein the autoimmune disorder is selected from the group consisting ofmultiple sclerosis, human systemic lupus, rheumatoid arthritis,inflammatory bowel disease, treating psoriasis, Type 1 diabetesmellitus, acute disseminated encephalomyelitis, Addison's disease,alopecia universalis, ankylosing spondylitisis, antiphospholipidantibody syndrome, aplastic anemia, autoimmune hemolytic anemia,autoimmune hepatitis, autoimmune inner ear disease, autoimmunelymphoproliferative syndrome, autoimmune oophoritis, Balo disease,Behcet's disease, bullous pemphigoid, cardiomyopathy, Chagas' disease,chronic fatigue immune dysfunction syndrome, chronic inflammatorydemyelinating polyneuropathy, Crohn's disease, cicatrical pemphigoid,coeliac sprue-dermatitis herpetiformis, cold agglutinin disease, CRESTsyndrome, Degos disease, discoid lupus, dysautonomia, endometriosis,essential mixed cryoglobulinemia, fibromyalgia-fibromyositis,Goodpasture's syndrome, Grave's disease, Guillain-Barre syndrome,Hashimoto's thyroiditis, Hidradenitis suppurativa, idiopathic and/oracute thrombocytopenic purpura, idiopathic pulmonary fibrosis, IgAneuropathy, interstitial cystitis, juvenile arthritis, Kawasaki'sdisease, lichen planus, Lyme disease, Meniere disease, mixed connectivetissue disease, myasthenia gravis, neuromyotonia, opsoclonus myoclonussyndrome, optic neuritis, Ord's thyroiditis, pemphigus vulgaris,pernicious anemia, polychondritis, polymyositis and dermatomyositis,primary biliary cirrhosis, polyarteritis nodosa, polyglandularsyndromes, polymyalgia rheumatica, primary agammaglobulinemia, Raynaudphenomenon, Reiter's syndrome, rheumatic fever, sarcoidosis,scleroderma, Sjögren's syndrome, stiff person syndrome, Takayasu'sarteritis, temporal arteritis, ulcerative colitis, uveitis, vasculitis,vitiligo, vulvodynia, and Wegener's granulomatosis.
 91. The method ofclaim 89, wherein the autoimmune disorder is scleroderma.
 92. The methodof claim 89, wherein the autoimmune disorder is multiple sclerosis. 93.The method of claim 89, wherein the autoimmune disorder is ulcerativecolitis.
 94. The method of claim 89, wherein the autoimmune disorder isChrohn's disease.
 95. The method of claim 89, wherein the autoimmunedisorder is Type 1 diabetes.
 96. The method of any one of claims 65-95,wherein the methods treats the disorder or cancer.
 97. A method ofdepleting a population of CD45+ cells, the method comprising contactingthe population with an effective amount of a conjugate represented bythe formula Ab-Am, wherein Ab is an antibody or antigen-binding fragmentthereof that binds CD45 and Am is an amatoxin.
 98. The method of claim97, wherein Am is represented by formula (IA)

wherein R₁ is H, OH, OR_(A), or OR_(C); R₂ is H, OH, OR_(B), or OR_(C);R_(A) and R_(B), together with the oxygen atoms to which they are bound,combine to form an optionally substituted 5-membered heterocyclolalkylgroup; R₃ is H, R_(C), or R_(D); R₄, R₅, R₆, and R₇ are eachindependently H, OH, OR_(C), OR_(D), R_(C), or R_(D); R₈ is OH, NH₂,OR_(C), OR_(D), NHR_(C), or NR_(C)R_(D); R₉ is H, OH, OR_(C), or OR_(D);X is —S—, —S(O)—, or —SO₂—; R_(C) is -L-Z; R_(D) is optionallysubstituted C₁-C₆ alkyl, optionally substituted C₁-C₆ heteroalkyl,optionally substituted C₂-C₆ alkenyl, optionally substituted C₂-C₆heteroalkenyl, optionally substituted C₂-C₆ alkynyl, optionallysubstituted C₂-C₆ heteroalkynyl, optionally substituted cycloalkyl,optionally substituted heterocycloalkyl, optionally substituted aryl, oroptionally substituted heteroaryl; L is optionally substituted C₁-C₆alkylene, optionally substituted C₁-C₆ heteroalkylene, optionallysubstituted C₂-C₆ alkenylene, optionally substituted C₂-C₆heteroalkenylene, optionally substituted C₂-C₆ alkynylene, optionallysubstituted C₂-C₆ heteroalkynylene, optionally substitutedcycloalkylene, optionally substituted heterocycloalkylene, optionallysubstituted arylene, or optionally substituted heteroarylene; and Z is achemical moiety formed from a coupling reaction between a reactivesubstituent present on L and a reactive substituent present within theantibody or antigen-binding fragment thereof, wherein Am comprisesexactly one R_(C) substituent.
 99. The method of claim 97, wherein Am isrepresented by formula (IB)

wherein R₁ is H, OH, OR_(A), or OR_(C); R₂ is H, OH, OR_(B), or OR_(C);R_(A) and R_(B), together with the oxygen atoms to which they are bound,combine to form an optionally substituted 5-membered heterocyclolalkylgroup; R₃ is H, R_(C), or R_(D); R₄, R₅, R₆, and R₇ are eachindependently H, OH, OR_(C), OR_(D), R_(C), or R_(D); R₈ is OH, NH₂,OR_(C), OR_(D), NHR_(C), or NR_(C)R_(D); R₉ is H, OH, OR_(C), or OR_(D);X is —S—, —S(O)—, or —SO₂—; R_(C) is -L-Z; R_(D) is optionallysubstituted C₁-C₆ alkyl, optionally substituted C₁-C₆ heteroalkyl,optionally substituted C₂-C₆ alkenyl, optionally substituted C₂-C₆heteroalkenyl, optionally substituted C₂-C₆ alkynyl, optionallysubstituted C₂-C₆ heteroalkynyl, optionally substituted cycloalkyl,optionally substituted heterocycloalkyl, optionally substituted aryl, oroptionally substituted heteroaryl; L is optionally substituted C₁-C₆alkylene, optionally substituted C₁-C₆ heteroalkylene, optionallysubstituted C₂-C₆ alkenylene, optionally substituted C₂-C₆heteroalkenylene, optionally substituted C₂-C₆ alkynylene, optionallysubstituted C₂-C₆ heteroalkynylene, optionally substitutedcycloalkylene, optionally substituted heterocycloalkylene, optionallysubstituted arylene, or optionally substituted heteroarylene; and Z is achemical moiety formed from a coupling reaction between a reactivesubstituent present on L and a reactive substituent present within theantibody or antigen-binding fragment thereof, wherein Am comprisesexactly one R_(C) substituent
 100. The method of claim 97, wherein thewherein the antibody or antigen-binding fragment thereof is conjugatedto the amatoxin by way of a cysteine residue in the Fc domain of theantibody or antigen-binding fragment thereof.
 101. The method of claim100, wherein the cysteine residue is introduced by way of a mutation inthe Fc domain of the antibody or antigen-binding fragment thereof. 102.The method of claim 101, wherein the cysteine residue is selected fromthe group consisting of Cys118, Cys239, and Cys265.
 103. The method ofclaim 100, wherein the cysteine residue is naturally occurring in the Fcdomain of the antibody or antigen-binding fragment thereof.
 104. Themethod of claim 103, wherein the Fc domain is an IgG Fc domain and thecysteine residue is selected from the group consisting of Cys261,Csy321, Cys367, and Cys425.
 105. The method of claim 98 or 99, whereinR₁ is H, OH, or OR_(A); R₂ is H, OH, or OR_(B); R_(A) and R_(B),together with the oxygen atoms to which they are bound, combine to form:

R₃, R₄, R₆, and R₇ are each H; R₅ is OR_(C); R₈ is OH or NH₂; and R₉ isH or OH.
 106. The method of claim 98 or 99, wherein R₁ and R₂ are eachindependently H or OH; R₃ is R_(C); R₄, R₆, and R₇ are each H; R₅ is H,OH, or OC₁-C₆ alkyl; R₈ is OH or NH₂; and R₉ is H or OH.
 107. The methodof claim 98 or 99, wherein R₁ and R₂ are each independently H or OH; R₃,R₆, and R₇ are each H; R₄ is OR_(C), or R_(C); R₅ is H, OH, or OC₁-C₆alkyl; R₈ is OH or NH₂; and R₉ is H or OH.
 108. The method of claim 98or 99, wherein R₁ and R₂ are each independently H or OH; R₃, R₆, and R₇are each H; R₄ and R₅ are each independently H or OH; R₈ is OR_(C) orNHR_(C); and R₉ is H or OH.
 109. The method of claim 97, wherein theantibody or antigen-binding fragment thereof is internalized by a CD45+cell.
 110. The method of claim 97, wherein the antibody orantigen-binding fragment thereof binds CD45 with a K_(d) of from about0.1 pM to about 1 μM.
 111. The method of claim 97, wherein the antibodyor antigen-binding fragment thereof binds CD45 with a k_(on) of fromabout 9×10⁻² M⁻¹ s⁻¹ to about 1×10² M⁻¹s⁻¹.
 112. The method of claim 97,wherein the antibody or antigen-binding fragment thereof competitivelyinhibits the binding of CD45 to a second antibody or antigen bindingfragment thereof, wherein the second antibody or antigen-bindingfragment thereof comprises the following complementarity determiningregions (CDRs): (SEQ ID NO: 16)a. a CDR-H1 having the amino acid sequence SYAMS; (SEQ ID NO: 17)b. a CDR-H2 having the amino acid sequence AISGSGGSTFYADSVRG;(SEQ ID NO: 18) c. a CDR-H3 having the amino acid sequence EVMGPIFFDY;(SEQ ID NO: 19) d. a CDR-L1 having the amino acid sequence RASQSIISSALA;(SEQ ID NO: 20) e. a CDR-L2 having the amino acid sequence GASSRAT; and(SEQ ID NO: 21) f. a CDR-L3 having the amino acid sequence QQYGSTPLT.


113. The method of claim 97, wherein the antibody or antigen-bindingfragment thereof is selected from the group consisting of a monoclonalantibody or antigen-binding fragment thereof, a polyclonal antibody orantigen-binding fragment thereof, a humanized antibody orantigen-binding fragment thereof, a bispecific antibody orantigen-binding fragment thereof, a dual-variable immunoglobulin domain,a single-chain Fv molecule (scFv), a diabody, a triabody, a nanobody, anantibody-like protein scaffold, a Fv fragment, a Fab fragment, a F(ab′)₂molecule, and a tandem di-scFV.
 114. A conjugate represented by theformula Ab-Am, wherein Ab is an antibody or antigen-binding fragmentthereof that binds CD45 and Am is an amatoxin.
 115. The conjugate ofclaim 114, wherein Am is represented by formula (IA)

wherein R₁ is H, OH, OR_(A), or OR_(C); R₂ is H, OH, OR_(B), or OR_(C);R_(A) and R_(B), together with the oxygen atoms to which they are bound,combine to form an optionally substituted 5-membered heterocyclolalkylgroup; R₃ is H, R_(C), or R_(D); R₄, R₅, R₆, and R₇ are eachindependently H, OH, OR_(C), OR_(D), R_(C), or R_(D); R₈ is OH, NH₂,OR_(C), OR_(D), NHR_(C), or NR_(C)R_(D); R₉ is H, OH, OR_(C), or OR_(D);X is —S—, —S(O)—, or —SO₂—; R_(C) is -L-Z; R_(D) is optionallysubstituted C₁-C₆ alkyl, optionally substituted C₁-C₆ heteroalkyl,optionally substituted C₂-C₆ alkenyl, optionally substituted C₂-C₆heteroalkenyl, optionally substituted C₂-C₆ alkynyl, optionallysubstituted C₂-C₆ heteroalkynyl, optionally substituted cycloalkyl,optionally substituted heterocycloalkyl, optionally substituted aryl, oroptionally substituted heteroaryl; L is optionally substituted C₁-C₆alkylene, optionally substituted C₁-C₆ heteroalkylene, optionallysubstituted C₂-C₆ alkenylene, optionally substituted C₂-C₆heteroalkenylene, optionally substituted C₂-C₆ alkynylene, optionallysubstituted C₂-C₆ heteroalkynylene, optionally substitutedcycloalkylene, optionally substituted heterocycloalkylene, optionallysubstituted arylene, or optionally substituted heteroarylene; and Z is achemical moiety formed from a coupling reaction between a reactivesubstituent present on L and a reactive substituent present within theantibody or antigen-binding fragment thereof, wherein Am comprisesexactly one R_(C) substituent.
 116. The conjugate of claim 114, whereinAm is represented by formula (IB)

wherein R₁ is H, OH, OR_(A), or OR_(C); R₂ is H, OH, OR_(B), or OR_(C);R_(A) and R_(B), together with the oxygen atoms to which they are bound,combine to form an optionally substituted 5-membered heterocyclolalkylgroup; R₃ is H, R_(C), or R_(D); R₄, R₅, R₆, and R₇ are eachindependently H, OH, OR_(C), OR_(D), R_(C), or R_(D); R₈ is OH, NH₂,OR_(C), OR_(D), NHR_(C), or NR_(C)R_(D); R₉ is H, OH, OR_(C), or OR_(D);X is —S—, —S(O)—, or —SO₂—; R_(C) is -L-Z; R_(D) is optionallysubstituted C₁-C₆ alkyl, optionally substituted C₁-C₆ heteroalkyl,optionally substituted C₂-C₆ alkenyl, optionally substituted C₂-C₆heteroalkenyl, optionally substituted C₂-C₆ alkynyl, optionallysubstituted C₂-C₆ heteroalkynyl, optionally substituted cycloalkyl,optionally substituted heterocycloalkyl, optionally substituted aryl, oroptionally substituted heteroaryl; L is optionally substituted C₁-C₆alkylene, optionally substituted C₁-C₆ heteroalkylene, optionallysubstituted C₂-C₆ alkenylene, optionally substituted C₂-C₆heteroalkenylene, optionally substituted C₂-C₆ alkynylene, optionallysubstituted C₂-C₆ heteroalkynylene, optionally substitutedcycloalkylene, optionally substituted heterocycloalkylene, optionallysubstituted arylene, or optionally substituted heteroarylene; and Z is achemical moiety formed from a coupling reaction between a reactivesubstituent present on L and a reactive substituent present within theantibody or antigen-binding fragment thereof, wherein Am comprisesexactly one R_(C) substituent
 117. The conjugate of claim 114, whereinthe antibody or antigen-binding fragment thereof is conjugated to theamatoxin by way of a cysteine residue in the Fc domain of the antibodyor antigen-binding fragment thereof.
 118. The conjugate of claim 117,wherein the cysteine residue is introduced by way of a mutation in theFc domain of the antibody or antigen-binding fragment thereof.
 119. Theconjugate of claim 118, wherein the cysteine residue is selected fromthe group consisting of Cys118, Cys239, and Cys265.
 120. The conjugateof claim 117, wherein the cysteine residue is naturally occurring in theFc domain of the antibody or antigen-binding fragment thereof.
 121. Theconjugate of claim 120, wherein the Fc domain is an IgG Fc domain andthe cysteine residue is selected from the group consisting of Cys261,Csy321, Cys367, and Cys425.
 122. The conjugate of claim 115 or 116,wherein R₁ is H, OH, or OR_(A); R₂ is H, OH, or OR_(B); R_(A) and R_(B),together with the oxygen atoms to which they are bound, combine to form:

R₃, R₄, R₆, and R₇ are each H; R₅ is OR_(C); R₈ is OH or NH₂; and R₉ isH or OH.
 123. The conjugate of claim 115 or 116, wherein R₁ and R₂ areeach independently H or OH; R₃ is R_(C); R₄, R₆, and R₇ are each H; R₅is H, OH, or OC₁-C₆ alkyl; R₈ is OH or NH₂; and R₉ is H or OH.
 124. Theconjugate of claim 115 or 116, wherein R₁ and R₂ are each independentlyH or OH; R₃, R₆, and R₇ are each H; R₄ is OR_(C), or R_(C); R₅ is H, OH,or OC₁-C₆ alkyl; R₈ is OH or NH₂; and R₉ is H or OH.
 125. The conjugateof claim 115 or 116, wherein R₁ and R₂ are each independently H or OH;R₃, R₆, and R₇ are each H; R₄ and R₅ are each independently H or OH; R₈is OR_(C) or NHR_(C); and R₉ is H or OH.
 126. The conjugate of claim114, wherein the antibody or antigen-binding fragment thereof isinternalized by a CD45+ cell.
 127. The conjugate of claim 114, whereinthe antibody or antigen-binding fragment thereof binds CD45 with a K_(d)of from about 0.1 pM to about 1 μM.
 128. The conjugate of claim 114,wherein the antibody or antigen-binding fragment thereof binds CD45 witha k_(on) of from about 9×10⁻² M⁻¹ s⁻¹ to about 1×10² M⁻¹s⁻¹.
 129. Theconjugate of claim 114, wherein the antibody or antigen-binding fragmentthereof competitively inhibits the binding of CD45 to a second antibodyor antigen binding fragment thereof, wherein the second antibody orantigen-binding fragment thereof comprises the following complementaritydetermining regions (CDRs): (SEQ ID NO: 16)a. a CDR-H1 having the amino acid sequence SYAMS; (SEQ ID NO: 17)b. a CDR-H2 having the amino acid sequence AISGSGGSTFYADSVRG;(SEQ ID NO: 18) c. a CDR-H3 having the amino acid sequence EVMGPIFFDY;(SEQ ID NO: 19) d. a CDR-L1 having the amino acid sequence RASQSIISSALA;(SEQ ID NO: 20) e. a CDR-L2 having the amino acid sequence GASSRAT; and(SEQ ID NO: 21) f. a CDR-L3 having the amino acid sequence QQYGSTPLT.


130. The conjugate of claim 114, wherein the antibody or antigen-bindingfragment thereof is selected from the group consisting of a monoclonalantibody or antigen-binding fragment thereof, a polyclonal antibody orantigen-binding fragment thereof, a humanized antibody orantigen-binding fragment thereof, a bispecific antibody orantigen-binding fragment thereof, a dual-variable immunoglobulin domain,a single-chain Fv molecule (scFv), a diabody, a triabody, a nanobody, anantibody-like protein scaffold, a Fv fragment, a Fab fragment, a F(ab′)₂molecule, and a tandem di-scFV.
 131. A method of depleting a populationof CD135+ cells, the method comprising contacting the population with aneffective amount of a conjugate represented by the formula Ab-Am,wherein Ab is an antibody or antigen-binding fragment thereof that bindsCD135 and Am is an amatoxin.
 132. The method of claim 131, wherein Am isrepresented by formula (IA)

wherein R₁ is H, OH, OR_(A), or OR_(C); R₂ is H, OH, OR_(B), or OR_(C);R_(A) and R_(B), together with the oxygen atoms to which they are bound,combine to form an optionally substituted 5-membered heterocyclolalkylgroup; R₃ is H, R_(C), or R_(D); R₄, R₅, R₆, and R₇ are eachindependently H, OH, OR_(C), OR_(D), R_(C), or R_(D); R₈ is OH, NH₂,OR_(C), OR_(D), NHR_(C), or NR_(C)R_(D); R₉ is H, OH, OR_(C), or OR_(D);X is —S—, —S(O)—, or —SO₂—; R_(C) is -L-Z; R_(D) is optionallysubstituted C₁-C₆ alkyl, optionally substituted C₁-C₆ heteroalkyl,optionally substituted C₂-C₆ alkenyl, optionally substituted C₂-C₆heteroalkenyl, optionally substituted C₂-C₆ alkynyl, optionallysubstituted C₂-C₆ heteroalkynyl, optionally substituted cycloalkyl,optionally substituted heterocycloalkyl, optionally substituted aryl, oroptionally substituted heteroaryl; L is optionally substituted C₁-C₆alkylene, optionally substituted C₁-C₆ heteroalkylene, optionallysubstituted C₂-C₆ alkenylene, optionally substituted C₂-C₆heteroalkenylene, optionally substituted C₂-C₆ alkynylene, optionallysubstituted C₂-C₆ heteroalkynylene, optionally substitutedcycloalkylene, optionally substituted heterocycloalkylene, optionallysubstituted arylene, or optionally substituted heteroarylene; and Z is achemical moiety formed from a coupling reaction between a reactivesubstituent present on L and a reactive substituent present within theantibody or antigen-binding fragment thereof, wherein Am comprisesexactly one R_(C) substituent.
 133. The method of claim 131, wherein Amis represented by formula (IB)

wherein R₁ is H, OH, OR_(A), or OR_(C); R₂ is H, OH, OR_(B), or OR_(C);R_(A) and R_(B), together with the oxygen atoms to which they are bound,combine to form an optionally substituted 5-membered heterocyclolalkylgroup; R₃ is H, R_(C), or R_(D); R₄, R₅, R₆, and R₇ are eachindependently H, OH, OR_(C), OR_(D), R_(C), or R_(D); R₈ is OH, NH₂,OR_(C), OR_(D), NHR_(C), or NR_(C)R_(D); R₉ is H, OH, OR_(C), or OR_(D);X is —S—, —S(O)—, or —SO₂—; R_(C) is -L-Z; R_(D) is optionallysubstituted C₁-C₆ alkyl, optionally substituted C₁-C₆ heteroalkyl,optionally substituted C₂-C₆ alkenyl, optionally substituted C₂-C₆heteroalkenyl, optionally substituted C₂-C₆ alkynyl, optionallysubstituted C₂-C₆ heteroalkynyl, optionally substituted cycloalkyl,optionally substituted heterocycloalkyl, optionally substituted aryl, oroptionally substituted heteroaryl; L is optionally substituted C₁-C₆alkylene, optionally substituted C₁-C₆ heteroalkylene, optionallysubstituted C₂-C₆ alkenylene, optionally substituted C₂-C₆heteroalkenylene, optionally substituted C₂-C₆ alkynylene, optionallysubstituted C₂-C₆ heteroalkynylene, optionally substitutedcycloalkylene, optionally substituted heterocycloalkylene, optionallysubstituted arylene, or optionally substituted heteroarylene; and Z is achemical moiety formed from a coupling reaction between a reactivesubstituent present on L and a reactive substituent present within theantibody or antigen-binding fragment thereof, wherein Am comprisesexactly one R_(C) substituent
 134. The method of claim 131, wherein thewherein the antibody or antigen-binding fragment thereof is conjugatedto the amatoxin by way of a cysteine residue in the Fc domain of theantibody or antigen-binding fragment thereof.
 135. The method of claim134, wherein the cysteine residue is introduced by way of a mutation inthe Fc domain of the antibody or antigen-binding fragment thereof. 136.The method of claim 135, wherein the cysteine residue is selected fromthe group consisting of Cys118, Cys239, and Cys265.
 137. The method ofclaim 134, wherein the cysteine residue is naturally occurring in the Fcdomain of the antibody or antigen-binding fragment thereof.
 138. Themethod of claim 137, wherein the Fc domain is an IgG Fc domain and thecysteine residue is selected from the group consisting of Cys261,Csy321, Cys367, and Cys425.
 139. The method of claim 132 or 133, whereinR₁ is H, OH, or OR_(A); R₂ is H, OH, or OR_(B); R_(A) and R_(B),together with the oxygen atoms to which they are bound, combine to form:

R₃, R₄, R₆, and R₇ are each H; R₅ is OR_(C); R₈ is OH or NH₂; and R₉ isH or OH.
 140. The method of claim 132 or 133, wherein R₁ and R₂ are eachindependently H or OH; R₃ is R_(C); R₄, R₆, and R₇ are each H; R₅ is H,OH, or OC₁-C₆ alkyl; R₈ is OH or NH₂; and R₉ is H or OH.
 141. The methodof claim 132 or 133, wherein R₁ and R₂ are each independently H or OH;R₃, R₆, and R₇ are each H; R₄ is OR_(C), or R_(C); R₅ is H, OH, orOC₁-C₆ alkyl; R₈ is OH or NH₂; and R₉ is H or OH.
 142. The method ofclaim 132 or 133, wherein R₁ and R₂ are each independently H or OH; R₃,R₆, and R₇ are each H; R₄ and R₅ are each independently H or OH; R₈ isOR_(C) or NHR_(C); and R₉ is H or OH.
 143. The method of claim 131,wherein the antibody or antigen-binding fragment thereof is internalizedby a CD135+ cell.
 144. The method of claim 131, wherein the antibody orantigen-binding fragment thereof binds CD135 with a K_(d) of from about0.1 pM to about 1 μM.
 145. The method of claim 131, wherein the antibodyor antigen-binding fragment thereof binds CD135 with a k_(on) of fromabout 9×10⁻² M⁻¹ s⁻¹ to about 1×10² M⁻¹s⁻¹.
 146. The method of claim131, wherein the antibody or antigen-binding fragment thereofcompetitively inhibits the binding of CD135 to a second antibody orantigen binding fragment thereof, wherein the second antibody orantigen-binding fragment thereof comprises the following complementaritydetermining regions (CDRs): (SEQ ID NO: 1)a. a CDR-H1 having the amino acid sequence SYYMH; (SEQ ID NO: 2)b. a CDR-H2 having the amino acid sequence IINPSGGSTSYAQKFQG;(SEQ ID NO: 3) c. a CDR-H3 having the amino acid sequence GVGAHDAFDI or(SEQ ID NO: 4) VVAAAVADY; (SEQ ID NO: 5)d. a CDR-L1 having the amino acid sequence RSSQSLLHSNGNNYLD or(SEQ ID NO: 6) RSSQSLLHSNGYNYLD; (SEQ ID NO: 7)e. a CDR-L2 having the amino acid sequence LGSNRAS; and (SEQ ID NO: 8)f. a CDR-L3 having the amino acid sequence MQGTHPAIS or (SEQ ID NO: 9)MQSLQTPFT.


147. The method of claim 131, wherein the antibody or antigen-bindingfragment thereof competitively inhibits the binding of CD135 to a secondantibody or antigen binding fragment thereof, wherein the secondantibody or antigen-binding fragment thereof comprises the followingcomplementarity determining regions (CDRs): (SEQ ID NO: 10)a. a CDR-H1 having the amino acid sequence SYAIS; (SEQ ID NO: 11)b. a CDR-H2 having the amino acid sequence GIIPIFGTANYAQKFQG;(SEQ ID NO: 12) c. a CDR-H3 having the amino acid sequenceFALFGFREQAFDI; (SEQ ID NO: 13)d. a CDR-L1 having the amino acid sequence RASQSISSYLN; (SEQ ID NO: 14)e. a CDR-L2 having the amino acid sequence AASSLQS; and (SEQ ID NO: 15)f. a CDR-L3 having the amino acid sequence QQSYSTPFT.


148. The method of claim 131, wherein the antibody or antigen-bindingfragment thereof is selected from the group consisting of a monoclonalantibody or antigen-binding fragment thereof, a polyclonal antibody orantigen-binding fragment thereof, a humanized antibody orantigen-binding fragment thereof, a bispecific antibody orantigen-binding fragment thereof, a dual-variable immunoglobulin domain,a single-chain Fv molecule (scFv), a diabody, a triabody, a nanobody, anantibody-like protein scaffold, a Fv fragment, a Fab fragment, a F(ab′)₂molecule, and a tandem di-scFV.
 149. A conjugate represented by theformula Ab-Am, wherein Ab is an antibody or antigen-binding fragmentthereof that binds CD135 and Am is an amatoxin.
 150. The conjugate ofclaim 149, wherein Am is represented by formula (IA)

wherein R₁ is H, OH, OR_(A), or OR_(C); R₂ is H, OH, OR_(B), or OR_(C);R_(A) and R_(B), together with the oxygen atoms to which they are bound,combine to form an optionally substituted 5-membered heterocyclolalkylgroup; R₃ is H, R_(C), or R_(D); R₄, R₅, R₆, and R₇ are eachindependently H, OH, OR_(C), OR_(D), R_(C), or R_(D); R₈ is OH, NH₂,OR_(C), OR_(D), NHR_(C), or NR_(C)R_(D); R₉ is H, OH, OR_(C), or OR_(D);X is —S—, —S(O)—, or —SO₂—; R_(C) is -L-Z; R_(D) is optionallysubstituted C₁-C₆ alkyl, optionally substituted C₁-C₆ heteroalkyl,optionally substituted C₂-C₆ alkenyl, optionally substituted C₂-C₆heteroalkenyl, optionally substituted C₂-C₆ alkynyl, optionallysubstituted C₂-C₆ heteroalkynyl, optionally substituted cycloalkyl,optionally substituted heterocycloalkyl, optionally substituted aryl, oroptionally substituted heteroaryl; L is optionally substituted C₁-C₆alkylene, optionally substituted C₁-C₆ heteroalkylene, optionallysubstituted C₂-C₆ alkenylene, optionally substituted C₂-C₆heteroalkenylene, optionally substituted C₂-C₆ alkynylene, optionallysubstituted C₂-C₆ heteroalkynylene, optionally substitutedcycloalkylene, optionally substituted heterocycloalkylene, optionallysubstituted arylene, or optionally substituted heteroarylene; and Z is achemical moiety formed from a coupling reaction between a reactivesubstituent present on L and a reactive substituent present within theantibody or antigen-binding fragment thereof, wherein Am comprisesexactly one R_(C) substituent.
 151. The conjugate of claim 149, whereinAm is represented by formula (IB)

wherein R₁ is H, OH, OR_(A), or OR_(C); R₂ is H, OH, OR_(B), or OR_(C);R_(A) and R_(B), together with the oxygen atoms to which they are bound,combine to form an optionally substituted 5-membered heterocyclolalkylgroup; R₃ is H, R_(C), or R_(D); R₄, R₅, R₆, and R₇ are eachindependently H, OH, OR_(C), OR_(D), R_(C), or R_(D); R₈ is OH, NH₂,OR_(C), OR_(D), NHR_(C), or NR_(C)R_(D); R₉ is H, OH, OR_(C), or OR_(D);X is —S—, —S(O)—, or —SO₂—; R_(C) is -L-Z; R_(D) is optionallysubstituted C₁-C₆ alkyl, optionally substituted C₁-C₆ heteroalkyl,optionally substituted C₂-C₆ alkenyl, optionally substituted C₂-C₆heteroalkenyl, optionally substituted C₂-C₆ alkynyl, optionallysubstituted C₂-C₆ heteroalkynyl, optionally substituted cycloalkyl,optionally substituted heterocycloalkyl, optionally substituted aryl, oroptionally substituted heteroaryl; L is optionally substituted C₁-C₆alkylene, optionally substituted C₁-C₆ heteroalkylene, optionallysubstituted C₂-C₆ alkenylene, optionally substituted C₂-C₆heteroalkenylene, optionally substituted C₂-C₆ alkynylene, optionallysubstituted C₂-C₆ heteroalkynylene, optionally substitutedcycloalkylene, optionally substituted heterocycloalkylene, optionallysubstituted arylene, or optionally substituted heteroarylene; and Z is achemical moiety formed from a coupling reaction between a reactivesubstituent present on L and a reactive substituent present within theantibody or antigen-binding fragment thereof, wherein Am comprisesexactly one R_(C) substituent
 152. The conjugate of claim 149, whereinthe wherein the antibody or antigen-binding fragment thereof isconjugated to the amatoxin by way of a cysteine residue in the Fc domainof the antibody or antigen-binding fragment thereof.
 153. The conjugateof claim 152, wherein the cysteine residue is introduced by way of amutation in the Fc domain of the antibody or antigen-binding fragmentthereof.
 154. The conjugate of claim 153, wherein the cysteine residueis selected from the group consisting of Cys118, Cys239, and Cys265.155. The conjugate of claim 152, wherein the cysteine residue isnaturally occurring in the Fc domain of the antibody or antigen-bindingfragment thereof.
 156. The conjugate of claim 155, wherein the Fc domainis an IgG Fc domain and the cysteine residue is selected from the groupconsisting of Cys261, Csy321, Cys367, and Cys425.
 157. The conjugate ofclaim 150 or 151, wherein R₁ is H, OH, or OR_(A); R₂ is H, OH, orOR_(B); R_(A) and R_(B), together with the oxygen atoms to which theyare bound, combine to form:

R₃, R₄, R₆, and R₇ are each H; R₅ is OR_(C); R₈ is OH or NH₂; and R₉ isH or OH.
 158. The conjugate of claim 150 or 151, wherein R₁ and R₂ areeach independently H or OH; R₃ is R_(C); R₄, R₆, and R₇ are each H; R₅is H, OH, or OC₁-C₆ alkyl; R₈ is OH or NH₂; and R₉ is H or OH.
 159. Theconjugate of claim 150 or 151, wherein R₁ and R₂ are each independentlyH or OH; R₃, R₆, and R₇ are each H; R₄ is OR_(C), or R_(C); R₅ is H, OH,or OC₁-C₆ alkyl; R₈ is OH or NH₂; and R₉ is H or OH.
 160. The conjugateof claim 150 or 151, wherein R₁ and R₂ are each independently H or OH;R₃, R₆, and R₇ are each H; R₄ and R₅ are each independently H or OH; R₈is OR_(C) or NHR_(C); and R₉ is H or OH.
 161. The conjugate of claim149, wherein the antibody or antigen-binding fragment thereof isinternalized by a CD135+ cell.
 162. The conjugate of claim 149, whereinthe antibody or antigen-binding fragment thereof binds CD135 with aK_(d) of from about 0.1 pM to about 1 μM.
 163. The conjugate of claim149, wherein the antibody or antigen-binding fragment thereof bindsCD135 with a k_(on) of from about 9×10⁻² M⁻¹ s⁻¹ to about 1×10² M⁻¹s⁻¹.164. The conjugate of claim 149, wherein the antibody or antigen-bindingfragment thereof competitively inhibits the binding of CD135 to a secondantibody or antigen binding fragment thereof, wherein the secondantibody or antigen-binding fragment thereof comprises the followingcomplementarity determining regions (CDRs): (SEQ ID NO: 1)a. a CDR-H1 having the amino acid sequence SYYMH; (SEQ ID NO: 2)b. a CDR-H2 having the amino acid sequence IINPSGGSTSYAQKFQG;(SEQ ID NO: 3) c. a CDR-H3 having the amino acid sequence GVGAHDAFDI or(SEQ ID NO: 4) VVAAAVADY; (SEQ ID NO: 5)d. a CDR-L1 having the amino acid sequence RSSQSLLHSNGNNYLD or(SEQ ID NO: 6) RSSQSLLHSNGYNYLD; (SEQ ID NO: 7)e. a CDR-L2 having the amino acid sequence LGSNRAS; and (SEQ ID NO: 8)f. a CDR-L3 having the amino acid sequence MQGTHPAIS or (SEQ ID NO: 9)MQSLQTPFT.


165. The conjugate of claim 149, wherein the antibody or antigen-bindingfragment thereof competitively inhibits the binding of CD135 to a secondantibody or antigen binding fragment thereof, wherein the secondantibody or antigen-binding fragment thereof comprises the followingcomplementarity determining regions (CDRs): (SEQ ID NO: 10)a. a CDR-H1 having the amino acid sequence SYAIS; (SEQ ID NO: 11)b. a CDR-H2 having the amino acid sequence GIIPIFGTANYAQKFQG;(SEQ ID NO: 12) c. a CDR-H3 having the amino acid sequenceFALFGFREQAFDI; (SEQ ID NO: 13)d. a CDR-L1 having the amino acid sequence RASQSISSYLN; (SEQ ID NO: 14)e. a CDR-L2 having the amino acid sequence AASSLQS; and (SEQ ID NO: 15)f. a CDR-L3 having the amino acid sequence QQSYSTPFT.


166. The conjugate of claim 149, wherein the antibody or antigen-bindingfragment thereof is selected from the group consisting of a monoclonalantibody or antigen-binding fragment thereof, a polyclonal antibody orantigen-binding fragment thereof, a humanized antibody orantigen-binding fragment thereof, a bispecific antibody orantigen-binding fragment thereof, a dual-variable immunoglobulin domain,a single-chain Fv molecule (scFv), a diabody, a triabody, a nanobody, anantibody-like protein scaffold, a Fv fragment, a Fab fragment, a F(ab′)₂molecule, and a tandem di-scFV.
 167. A conjugate represented by theformula Ab-Cy, wherein Ab is an antibody or antigen-binding fragmentthereof that binds CD45 and Cy is pseudomonas exotoxin A.
 168. Aconjugate represented by the formula Ab-Cy, wherein Ab is an antibody orantigen-binding fragment thereof that binds CD45 and Cy is deBouganin.169. A conjugate represented by the formula Ab-Cy, wherein Ab is anantibody or antigen-binding fragment thereof that binds CD45 and Cy isdiphtheria toxin.
 170. A conjugate represented by the formula Ab-Cy,wherein Ab is an antibody or antigen-binding fragment thereof that bindsCD45 and Cy is saporin.
 171. A conjugate represented by the formulaAb-Cy, wherein Ab is an antibody or antigen-binding fragment thereofthat binds CD45 and Cy is maytansine or a maytansinoid.
 172. A conjugaterepresented by the formula Ab-Cy, wherein Ab is an antibody orantigen-binding fragment thereof that binds CD45 and Cy is anauristatin.
 173. A conjugate represented by the formula Ab-Cy, whereinAb is an antibody or antigen-binding fragment thereof that binds CD45and Cy is an anthracycline.
 174. A conjugate represented by the formulaAb-Cy, wherein Ab is an antibody or an antigen-binding fragment thereofthat binds CD45 and Cy is a calicheamicin.
 175. A conjugate representedby the formula Ab-Cy, wherein Ab is an antibody or an antigen-bindingfragment thereof that binds CD45 and Cy is irinotecan.
 176. A conjugaterepresented by the formula Ab-Cy, wherein Ab is an antibody or anantigen-binding fragment thereof that binds CD45 and Cy is SN-38.
 177. Aconjugate represented by the formula Ab-Cy, wherein Ab is an antibody oran antigen-binding fragment thereof that binds CD45 and Cy is aduocarmycin.
 178. A conjugate represented by the formula Ab-Cy, whereinAb is an antibody or an antigen-binding fragment thereof that binds CD45and Cy is a pyrrolobenzodiazepine or a pyrrolobenzodiazepine dimer. 179.A conjugate represented by the formula Ab-Cy, wherein Ab is an antibodyor an antigen-binding fragment thereof that binds CD45 and Cy is anindolinobenzodiazepine or an indolinobenzodiazepine dimer.
 180. Aconjugate represented by the formula Ab-Cy, wherein Ab is an antibody orantigen-binding fragment thereof that binds CD135 and Cy is pseudomonasexotoxin A.
 181. A conjugate represented by the formula Ab-Cy, whereinAb is an antibody or antigen-binding fragment thereof that binds CD135and Cy is deBouganin.
 182. A conjugate represented by the formula Ab-Cy,wherein Ab is an antibody or antigen-binding fragment thereof that bindsCD135 and Cy is diphtheria toxin.
 183. A conjugate represented by theformula Ab-Cy, wherein Ab is an antibody or antigen-binding fragmentthereof that binds CD135 and Cy is saporin.
 184. A conjugate representedby the formula Ab-Cy, wherein Ab is an antibody or antigen-bindingfragment thereof that binds CD135 and Cy is maytansine or amaytansinoid.
 185. A conjugate represented by the formula Ab-Cy, whereinAb is an antibody or antigen-binding fragment thereof that binds CD135and Cy is an auristatin.
 186. A conjugate represented by the formulaAb-Cy, wherein Ab is an antibody or antigen-binding fragment thereofthat binds CD135 and Cy is an anthracycline.
 187. A conjugaterepresented by the formula Ab-Cy, wherein Ab is an antibody or anantigen-binding fragment thereof that binds CD135 and Cy is acalicheamicin.
 188. A conjugate represented by the formula Ab-Cy,wherein Ab is an antibody or an antigen-binding fragment thereof thatbinds CD135 and Cy is irinotecan.
 189. A conjugate represented by theformula Ab-Cy, wherein Ab is an antibody or an antigen-binding fragmentthereof that binds CD135 and Cy is SN-38.
 190. A conjugate representedby the formula Ab-Cy, wherein Ab is an antibody or an antigen-bindingfragment thereof that binds CD135 and Cy is a duocarmycin.
 191. Aconjugate represented by the formula Ab-Cy, wherein Ab is an antibody oran antigen-binding fragment thereof that binds CD135 and Cy is apyrrolobenzodiazepine or a pyrrolobenzodiazepine dimer.
 192. A conjugaterepresented by the formula Ab-Cy, wherein Ab is an antibody or anantigen-binding fragment thereof that binds CD135 and Cy is anindolinobenzodiazepine or an indolinobenzodiazepine dimer.
 193. Aconjugate represented by the formula Ab-Cy, wherein Ab is an antibody orantigen-binding fragment thereof that binds CD34 and Cy is pseudomonasexotoxin A.
 194. A conjugate represented by the formula Ab-Cy, whereinAb is an antibody or antigen-binding fragment thereof that binds CD34and Cy is deBouganin.
 195. A conjugate represented by the formula Ab-Cy,wherein Ab is an antibody or antigen-binding fragment thereof that bindsCD34 and Cy is diphtheria toxin.
 196. A conjugate represented by theformula Ab-Cy, wherein Ab is an antibody or antigen-binding fragmentthereof that binds CD34 and Cy is saporin.
 197. A conjugate representedby the formula Ab-Cy, wherein Ab is an antibody or antigen-bindingfragment thereof that binds CD34 and Cy is maytansine or a maytansinoid.198. A conjugate represented by the formula Ab-Cy, wherein Ab is anantibody or antigen-binding fragment thereof that binds CD34 and Cy isan auristatin.
 199. A conjugate represented by the formula Ab-Cy,wherein Ab is an antibody or antigen-binding fragment thereof that bindsCD34 and Cy is an anthracycline.
 200. A conjugate represented by theformula Ab-Cy, wherein Ab is an antibody or an antigen-binding fragmentthereof that binds CD34 and Cy is a calicheamicin.
 201. A conjugaterepresented by the formula Ab-Cy, wherein Ab is an antibody or anantigen-binding fragment thereof that binds CD34 and Cy is irinotecan.202. A conjugate represented by the formula Ab-Cy, wherein Ab is anantibody or an antigen-binding fragment thereof that binds CD34 and Cyis SN-38.
 203. A conjugate represented by the formula Ab-Cy, wherein Abis an antibody or an antigen-binding fragment thereof that binds CD34and Cy is a duocarmycin.
 204. A conjugate represented by the formulaAb-Cy, wherein Ab is an antibody or an antigen-binding fragment thereofthat binds CD34 and Cy is a pyrrolobenzodiazepine or apyrrolobenzodiazepine dimer.
 205. A conjugate represented by the formulaAb-Cy, wherein Ab is an antibody or an antigen-binding fragment thereofthat binds CD34 and Cy is an indolinobenzodiazepine or anindolinobenzodiazepine dimer.
 206. A conjugate represented by theformula Ab-Cy, wherein Ab is an antibody or antigen-binding fragmentthereof that binds CD90 and Cy is pseudomonas exotoxin A.
 207. Aconjugate represented by the formula Ab-Cy, wherein Ab is an antibody orantigen-binding fragment thereof that binds CD90 and Cy is deBouganin.208. A conjugate represented by the formula Ab-Cy, wherein Ab is anantibody or antigen-binding fragment thereof that binds CD90 and Cy isdiphtheria toxin.
 209. A conjugate represented by the formula Ab-Cy,wherein Ab is an antibody or antigen-binding fragment thereof that bindsCD90 and Cy is saporin.
 210. A conjugate represented by the formulaAb-Cy, wherein Ab is an antibody or antigen-binding fragment thereofthat binds CD90 and Cy is maytansine or a maytansinoid.
 211. A conjugaterepresented by the formula Ab-Cy, wherein Ab is an antibody orantigen-binding fragment thereof that binds CD90 and Cy is anauristatin.
 212. A conjugate represented by the formula Ab-Cy, whereinAb is an antibody or antigen-binding fragment thereof that binds CD90and Cy is an anthracycline.
 213. A conjugate represented by the formulaAb-Cy, wherein Ab is an antibody or an antigen-binding fragment thereofthat binds CD90 and Cy is a calicheamicin.
 214. A conjugate representedby the formula Ab-Cy, wherein Ab is an antibody or an antigen-bindingfragment thereof that binds CD90 and Cy is irinotecan.
 215. A conjugaterepresented by the formula Ab-Cy, wherein Ab is an antibody or anantigen-binding fragment thereof that binds CD90 and Cy is SN-38.
 216. Aconjugate represented by the formula Ab-Cy, wherein Ab is an antibody oran antigen-binding fragment thereof that binds CD90 and Cy is aduocarmycin.
 217. A conjugate represented by the formula Ab-Cy, whereinAb is an antibody or an antigen-binding fragment thereof that binds CD90and Cy is a pyrrolobenzodiazepine or a pyrrolobenzodiazepine dimer. 218.A conjugate represented by the formula Ab-Cy, wherein Ab is an antibodyor an antigen-binding fragment thereof that binds CD90 and Cy is anindolinobenzodiazepine or an indolinobenzodiazepine dimer.
 219. Aconjugate represented by the formula Ab-Cy, wherein Ab is an antibody orantigen-binding fragment thereof that binds CD110 and Cy is pseudomonasexotoxin A.
 220. A conjugate represented by the formula Ab-Cy, whereinAb is an antibody or antigen-binding fragment thereof that binds CD110and Cy is deBouganin.
 221. A conjugate represented by the formula Ab-Cy,wherein Ab is an antibody or antigen-binding fragment thereof that bindsCD110 and Cy is diphtheria toxin.
 222. A conjugate represented by theformula Ab-Cy, wherein Ab is an antibody or antigen-binding fragmentthereof that binds CD110 and Cy is saporin.
 223. A conjugate representedby the formula Ab-Cy, wherein Ab is an antibody or antigen-bindingfragment thereof that binds CD110 and Cy is maytansine or amaytansinoid.
 224. A conjugate represented by the formula Ab-Cy, whereinAb is an antibody or antigen-binding fragment thereof that binds CD110and Cy is an auristatin.
 225. A conjugate represented by the formulaAb-Cy, wherein Ab is an antibody or antigen-binding fragment thereofthat binds CD110 and Cy is an anthracycline.
 226. A conjugaterepresented by the formula Ab-Cy, wherein Ab is an antibody or anantigen-binding fragment thereof that binds CD110 and Cy is acalicheamicin.
 227. A conjugate represented by the formula Ab-Cy,wherein Ab is an antibody or an antigen-binding fragment thereof thatbinds CD110 and Cy is irinotecan.
 228. A conjugate represented by theformula Ab-Cy, wherein Ab is an antibody or an antigen-binding fragmentthereof that binds CD110 and Cy is SN-38.
 229. A conjugate representedby the formula Ab-Cy, wherein Ab is an antibody or an antigen-bindingfragment thereof that binds CD110 and Cy is a duocarmycin.
 230. Aconjugate represented by the formula Ab-Cy, wherein Ab is an antibody oran antigen-binding fragment thereof that binds CD110 and Cy is apyrrolobenzodiazepine or a pyrrolobenzodiazepine dimer.
 231. A conjugaterepresented by the formula Ab-Cy, wherein Ab is an antibody or anantigen-binding fragment thereof that binds CD110 and Cy is anindolinobenzodiazepine or an indolinobenzodiazepine dimer.